New chemical entities are desperately needed that overcome the limitations of existing drugs for neglected diseases. Screening a diverse library of 10,000 drug-like compounds against 7 neglected disease pathogens resulted in an integrated dataset of 744 hits. We discuss the prioritization of these hits for each pathogen and the strong correlation observed between compounds active against more than two pathogens and mammalian cell toxicity. Our work suggests that the efficiency of early drug discovery for neglected diseases can be enhanced through a collaborative, multi-pathogen approach. The search for new drugs for human neglected diseases accelerated in the past decade, based on the recognition that addressing these infections was necessary for global poverty reduction. The expansion of discovery and development programmes was supported by donor investment, increasing participation of the industry and the creation of Product Development Partnership (PDP) enterprises. Despite these efforts, major discovery gaps remain as, apart from some repurposed drugs and a few new molecules for malaria, no new candidate has been recently transitioned from discovery into development for the major Neglected Tropical Diseases (NTDs). In this publication, we present a collaborative network model for drug discovery based on coordinated North-South partnerships. This network carried out low-to-medium throughput whole-organism screening assays against seven NTDs  together with an early assessment of compound toxicity in mammalian cells. We describe a screening campaign of 10,000 molecules, its outcome and the implications of this strategy for enhancing the efficiency and productivity of drug discovery for NTDs. The search for new antiparasitic drugs for use in humans has accelerated in the past decade, based partly on the growing recognition that addressing these widespread infections is necessary for poverty reduction. There is a consensus that the drugs available for these pathogens are far from optimal, plagued by susceptibility to resistance, lack of activity against key species (or stages of the life cycle), lack of adequate efficacy in field-compatible delivery regimens, and reliant on single agents for control programmes WHO-TDR has a long history of drug discovery and development for NTDs Despite these efforts, major gaps in the discovery of new chemical entities for neglected diseases remain, and apart from some repurposed drugs and few new molecules for malaria Plasmodium falciparum, Leishmania infantum, Trypanosoma brucei, Trypanosoma cruzi, Schistosoma mansoni, Onchocerca lienalis or O ochengi  and Brugia malayi. Higher-throughput methods have been developed for whole organism antimalarial and anti-Chagas screens, both in industrial and non-industrial settings A network model for drug discovery against multiple neglected diseases has been described as a suitable mechanism to overcome these challenges in vitro test is fully predictive of in vivo activity, the TDR strategy is generally accepted as standard practice in neglected diseases screens TDR has taken advantage of the availability of multiple assays available within its network to implement coordinated screening of highly triaged small-to-medium sized libraries against multiple organisms, combined with a rapid assessment of mammalian cell toxicity. Although no Here we present the outcome of screening a set of 10,000 compounds against seven (7) NTD pathogens in whole-organism assays through a collaborative network model. The implications of this approach, which we refer to as an integrated multi-pathogen screening strategy, for enhancing drug discovery productivity and efficiency for neglected diseases are discussed.A library of 10,000 compounds was purchased from Life Chemicals (Ukraine) after extensive review and analysis by medicinal chemists of over 50,000 available diverse structures. The key criteria used in the selection of molecules to be screened are diversity and novelty. To ensure a degree of diversity, selection was enhanced by using the computational informatics tool  in vitro and for efficacy in animal models The compound screening logistics and framework, including sampling, distribution to various screening centers and screening cascade for the various parasites are shown on P. falciparum, amphotericin B for L. infantum, nifurtimox for T cruzi, suramin and pentamidine for T. brucei, praziquantel for S. mansoni, immiticide, amocarzine and ivermectin for O. lienalis or O. ochengi, and ivermectin and diethylcarbamazine for B. malayi.Appropriate reference drugs, obtained either from Sigma or WHO-TDR collection, were used as positive controls P. falciparum K1 strain  T. brucei Squib 427 strain  T. cruzi Tulahuen CL2, beta- galactosidase strain (Chagas' Disease) L. infantum MHOM/MA(BE)/67 (leishmaniasis) S. mansoni Puerto Rican strain (schistosomiasis) B. malayi (lymphatic filariasis) O. ochengi or O. lienalis (onchocerciasis) Onchocerca volvulus, but due to lack of a suitable in vitro assay or animal models for this pathogen, O. ochengi and lienalis, which are both animal pathogens, are used in the primary and secondary assays The compound set was tested against the following parasites: intra-erythrocytic forms in human red blood cells of In some cases, particularly for the antifilarial screens, a second whole organism test with adult stage parasites is included prior to animal evaluation. The decision to utilize microfilariae for primary screening was based on throughput and biological considerations. It is difficult to acquire enough adult filariae to enable screening more than 1000 compounds per year L. infantum and T. cruzi, which include murine peritoneal macrophage host cells SV2 human lung fibroblast cells, respectively, in the culture system.An important part of the screen is the evaluation of all compounds for cytotoxicity using the diploid human embryonic lung fibroblast MRC-5 cell line . FurtherHits identified from the screens were initially compared with cytotoxicity data for selectivity. Further filtering of the data was done using the published ‘Hit’ criteria for all pathogens combined with classical medicinal chemistry criteria From the screen of 10,000 compounds, 744 were identified as active against at least one pathogen . Cytotox50<0.5 µg/ml for P. falciparum50 values of ∼0.15 µg/ml is structurally related to, but nonetheless distinct from, a series with activity against T. cruzi and T. brucei in vitro . One singleton [structure # 15] is structurally related to a series with activity against L. infantum [structure # 38] and the other was distinguished by a notable lack of activity with close analogs tested in this assay and therefore is of interest [structure # 16].33 of the 744 active compounds met the potency criteria of IC50<0.5 µg/ml for bloodstream forms of T. brucei Squib 427 strain. Of these, 67 compounds exemplified in [structures #1 to 6] were removed from further consideration due to poor selectivity vis-à-vis mammalian cells. An additional 7 compounds were discarded based on undesirable chemical attributes [structure # 39]. Of the remaining 31 molecules, TDR84116 , showed exceptional potency (IC50 = 0.08 µg/ml) against T. brucei and was later reconfirmed with a similar potency  against T. brucei rhodesiense (STIB-900). The remaining 20 actives represent 6 structural series and 11 singletons. All were less potent than TDR84116, with many showing IC50 values near the cut-off point. Some of these series are also active against T. cruzi . One series was prioritized for the onchocerciasis program (data not shown), while a third series appears interesting for a joint HAT/Chagas project  for poor selectivity versus mammalian cells (SI<50) and an additional 11 due to undesirable chemical attributes. The remaining 51 compounds were sorted into 10 series (45 compounds) and 6 singletons . Two of these series with excellent potency against T. cruzi, limited activity against other protozoa and SI values >50 (not shown), have been transferred to DNDi for further evaluation.171 of the 744 actives passed the initial criterion of ICin vivo.A series of 2-aminothiazole derivatives [structure # 23] was transferred to the University of Sao Paolo for further exploration, though potency and selectivity were lower than those observed for the series transferred to DNDi. A series with a benzo(thiazol-2-yl)acetamide pharmacophore  included 14 molecules with activity at concentrations as low as 0.22 µg/ml with an interesting pattern of selectivity against other pathogens. However, some showed limited selectivity against mammalian cells. Additional investment of medicinal chemistry should be made to clarify the potential for selectivity among the various parasites and especially with regard to mammalian cells, prior to analysis 50<2 µg/ml against L. infantum amastigotes in macrophages. Of these, 30 [structure # 25] were discarded for poor selectivity against mammalian cells (SI<20) and another 8 for undesirable chemical attributes . The remaining 77 compounds were organized in 11 series (58 compounds) and 22 singletons. In addition to the series described above for P. falciparum [structure # 16], several series are considered high priority based on potency (IC50<0.5 µg/ml in ≥2 compounds) and SI values >30, as well as on preliminary SAR and chemical considerations. One series , includes 17 molecules with no previous evidence of antiparasitic applications and, for most of the molecules, a clean selectivity profile against tested pathogens. Two other high interest series with respectively 9 and 6 analogues are represented by structures #42 and #43.115 compounds met the criterion of ICS. mansoni. 6144 of these were tested in a primary larval assay S. mansoni: . The 65 compounds were sorted by chemical attractiveness, potency against adult schistosomes in culture, selectivity against other parasites, and toxicity to mammalian cells in culture. Based on this analysis, 5 series encompassing 13 compounds were chosen for advancement through the African Network for Drug and Diagnostic Innovation (ANDI) 50<2 µg/ml . Further analysis of these series, including confirmation, titration, testing of additional analogs and assays of in vivo activity in mouse models of schistosomiasis, is needed to prove that any has sufficient merit to warrant further development.A subset of 9478 of the 10,000 compound collection was tested against the Puerto Rican strain of B. malayi, including 6063 against microfilariae and 756 against adult worms (these sets overlap). Activity criteria were 100% loss of motility at 10 µM for either stage. Greatest priority was given to compounds with activity against adult worms, as no macrofilaricide is available for use in control campaigns and the activity of ivermectin as a microfilaricidal agent is unlikely to be bettered in a new series. Of the test set, 53 compounds met the activity criterion for microfilariae (100% motility reduction) and an additional 58 inhibited adult worm motility ≥75% at 10 µM. These were titrated to determine an IC50 value to support prioritization. Following cytotoxicity triage , eight compounds met the activity criterion 50 value were also considered. Of these, the most interesting was TDR76699 [structure # 29]; this compound has very good activity against adult worms (IC50 = 0.1 µg/ml) and is also active against microfilariae (IC50 = 0.7 µg/ml). None of the other compounds tested against adult parasites had IC50 values ≤0.5 µg/ml, a threshold which would reduce concerns about specificity. However, a few series , appear interesting for future medicinal chemistry projects.As result of logistics issues attributed to lost shipments of some compounds, only 6160 molecules of the 10,000 compound set were tested, at a uniform molarity O. ochengi at the University of Buea in Cameroon, while a subset of 5088 compounds was successfully screened in UK by the Northwick Park Institute for Medical Research against O. lienalis, at a uniform molarity O. lienalis and 330 were active against microfilaria of O. ochengi (100% inhibition of motility at 12.5 µM). After removing molecules that were toxic to mammalian cells (IC50<5 µg/ml), 292 molecules remained for further evaluation. It is interesting to note that of the 330 O. ochengi microfilaria positives, 48 have already been found to also have full to moderate activity on the adult worms. From the latter sub-set, 3 distinct chemical series and one singleton [structure # 35] were active against both microfilaria and adults. One series  was deemed not interesting due to lack of selectivity against mammalian cells. The remaining series is being transferred to ANDI for further progression as part of a new collaboration.4240 compound s were screened against www.tdrtargets.orgIn summary, of the hits that survived toxicity triage, at least four series covering malaria, schistosomiasis, Chagas' disease and human African trypanosomiasis have been transferred to external partners  for further exploration of SAR, whilst promising series for leishmaniasis, schistosomiasis and onchocerciasis are being transferred to ANDI. Details, including structures of hits for malaria, HAT, Chagas' Disease and LF, that are not restricted by any legal agreements, are available through the TDR target database - Historically, antiparasitic drug discovery has relied on whole organism screens, as exemplified by recent reports of promising leads from whole-malaria parasite screens P. falciparum. This is due to the nature of the compound set and the fact that most known actives against P. falciparum were eliminated from the library. However, one of the identified novel malaria hit series is structurally similar to a previously identified series undergoing lead optimization in collaboration with Merck Serono (data not shown). This screening strategy also highlights the critical importance and value of early cytotoxicity assessment  is suggestive of a specific mechanism of action. Although azoles may be active against different parasites, they probably act by different mechanisms in each Our results also show that none of the anthelmintic hit series which showed no appreciable cytotoxicity was active on > one helminth pathogen, i.e., identified positives are selectively active in the schistosomiasis, onchocerciasis or LF screens [The major benefit of the current approach centers on the synergy gained by simultaneously screening a compound collection against multiple organisms. Salient advantages include:Compound handling can be better coordinated and organized to gain efficiency in the distribution process.Interpretation and analysis of data can be done in a coordinated manner, so that decisions about compound progression can be integrated with programmatic priorities for the various indications.Areas of greatest need can be balanced with quality of hits  to produce a portfolio of leads that offers the best chance for making significant advances in their respective area.It provides early validation of and confidence in hits identified and prioritized for the various pathogens.It helps to rapidly share lessons and create value around a set of compounds due to the significant amount of data collected from the multiple screens.www.dndi.org). It is therefore remarkable to see the interesting hit rate found in our screening campaign. The fact that we discovered different compound series, containing up to 17 similar molecules active against L. infantum, gives some degree of confidence to the results. This again points to the usefulness of careful selection of chemical libraries used for screening.It should be emphasized that it has been traditionally difficult to generate novel, robust hits against leishmaniasis, leaving the current screening and drug discovery pipeline weak for this disease . Finally, the screens do not always employ the most important target stage of the parasite . The screens are thus compromises between ideal and practical operations, with the central assumption inherent in this strategy: that drugs with broad-spectrum activity within a parasite group will be discovered and are of the greatest interest. On the logistics side: the network screening model requires careful plate formatting and repartitioning to minimize risks of inability to trace inter or intra screening variations.While the integrated screens presented here represent current “best practice”, many unknowns plague the screening pathways for new antiparasitic agents for human use. The dearth of safe and effective drugs for most NTDs means that it is difficult to fully validate the screening streams. Some of the most important antiparasitic drugs seem to require an interaction with the host immune system, for example, praziquantel for schistosomiasis and ivermectin for filariasis www.tdrtargets.org), a global open source database of molecular targets and drug like compounds that supports open innovation for infectious tropical diseases The integrated, multi-pathogen, collaborative approach to the discovery of new chemical entities described here promises to increase efficiency while reducing costs and second-stage attrition in the discovery of high-quality leads compared to operations which target pathogens individually. Although this work focuses on the initial whole organism pathogen screening programme for hit identification, it should be stressed that it is part of an integrated North-South drug discovery platform that includes medicinal chemistry, pharmacokinetics and early toxicology Table S1Results table.(DOC)Click here for additional data file.


While the impact of volatile anaesthetics to induce malignant hyperthermia (MH) is abundantly clear, the role of succinylcholine still remains controversial. To evaluate the influence of succinylcholine on porcine MH events, the authors investigated the hemodynamic and metabolic responses in MH susceptible (MHS) and non-susceptible (MHN) swine following either succinylcholine or halothane application alone or a combination of both substances.2 measurement were inserted into the femoral vein and the triceps muscle. Group A received succinylcholine 4 mg/kg, group B incremental doses of halothane  and group C succinylcholine and halothane simultaneously. Vital signs were recorded continuously.With approval of the local animal care committee 27 MHS and 30 MHN pigs were anaesthetized and mechanically ventilated. Fiberoptic probes for continuous PCOPrior to drug application measured values did not differ between MHS and MHN. While MHN pigs did not show relevant alterations, succinylcholine, halothane and the combination of both lead to significant hemodynamic and metabolic changes in MHS swine.Hemodynamic and metabolic alterations following succinylcholine were similar to halothane in MHS pigs. The combination of both pharmacological agents potentiated the observed effects. According to these results succinylcholine acted as an independent and supportive factor during onset of an MH episode. In predisposed humans and animals exposure to triggering agents may lead to a hypermetabolic muscular syndrome. Uncontrolled sarcoplasmic calcium release caused by mutations in the ryanodine receptor subtype 1 or in the dihydrophyridine receptor results in hypoxemia, hypercapnia, tachycardia, muscular rigidity, acidosis, hyperkalaemia and hyperthermia . For insin vitro, whereasin vitro. Furtherin vitro.Hence, the aim of the present study was to investigate the hemodynamic and metabolic response in MH susceptible (MHS) and non-susceptible (MHN) pigs following application of SCh, halothane or the combination of both pharmacological agents, to evaluate the influence of SCh on porcine MH events.With approval of the local animal care committee , 57 pietrain pigs weighing 30.6 (27.8 – 31.5) kg were examined. Prior to the investigation, MH susceptibility or wild type was determined by DNA analysis regarding the presence of a homozygous arginine 615 mutation of the ryanodine receptor indicating MHS.2) of 30–35 mmHg . Afterwards, two PCO2 probes  were placed under ultrasound guidance into the left triceps muscle and into the left femoral vein of the animals. Vital signs of the animals were monitored continuously by mean arterial blood pressure (MAP) in the saphenous artery, peripheral oxygen saturation (SaO2), ECG and rectal temperature. A warming blanket and an infrared lamp were used to prevent the animals from hypothermia. In 15 minutes intervals arterial blood samples were obtained and immediately analysed for pH, arterial oxygen partial pressure (PaO2), carbon dioxide partial pressure (PaCO2), base excess (BE) and lactate. Throughout the experiment 4 mL/kg/h Ringer solution  was administered via a central venous line placed in the right jugular vein.Anaesthesia was induced intravenously via an ear vein with thiopental (14 – 17 mg/kg) and maintained using a midazolam (0.2 - 0.4 mg/kg/h) and fentanyl (0.01 - 0.04 mg/kg/h) infusion in all swine. After the trachea was intubated  without the use of muscle relaxants, ventilator settings  were adjusted to keep an end-tidal carbon dioxide partial pressure  received two single boluses of SCh 4 mg/kg  at intervals of 15 minutes, while in the halothane group  the anaesthetic vapor setting was adjusted to achieve an end-tidal halothane concentration of 0.5 vol% for the first 15 minutes, followed by an elevation to 1 vol% for further 15 minutes. In a third group  a single bolus of SCh 4 mg/kg combined with an end-tidal halothane concentration of 0.5 vol% was applied at first and was augmented after 15 minutes by another SCh bolus 4 mg/kg and an increase of halothane to 1 vol%. Different sizes of the investigated groups were caused by the availability of the animals. Intramuscular and femoral venous PCO2 sensor consisted of an optode, kept in a heparin-coated microporous polyethylene tube of approximately 0.5 mm in diameter and permeable to the substance to be measured. The cylindrical sensor construction allowed measurements over the entire probe surface. Prior to insertion in the target tissue, the probes were calibrated in vitro by exposure to different standard gas concentrations .The fiberoptic PCOData are displayed as median and interquartile range. Statistical comparisons between and within the investigated groups were performed by repeated measures ANOVA and post-hoc Tukey test. A value of p < 0.05 was considered statistically significant.According to the predefined metabolic parameters none of the MHN swine developed any clinical signs of an MH episode after application of SCh, halothane or both agents. In contrast, MH occurred in all MHS animals of the halothane and the SCh plus halothane group, while only four out of nine MHS pigs in the SCh group fulfilled the defined criteria.2 were observed in all MHS animals. However, SCh did not induce a rise of body temperature in the MHS pigs.Prior to drug application hemodynamic variables and rectal temperature were comparable between the investigated groups. While in MHN swine SCh had little or no effect on hemodynamic parameters, relevant hypotension, tachycardia and an increase in end-tidal PCO2 only became significant in MHS swine. But, neither heart rate nor rectal temperatures were significantly affected by halothane application in normal or susceptible animals.Interestingly, incremental concentrations of halothane caused a significant decrease of arterial blood pressure in both investigated groups, while increases of end-tidal PCO2 were significantly different between susceptible and normal animals. It is noteworthy, that eleven of the twelve MHS pigs immediately died after reinjection of SCh and raise of end-tidal halothane concentration to 1 vol%. Even MHN pigs developed significant hypotension and tachycardia after administration of halothane plus SCh  with SCh induced MH crisis in absence of any inhalation anaesthetic were identified [In contrast, the underlying mode of action or the impact of SCh to elicit an MH remained unclear so far. Larach and colleagues rated 284 cases that were suspicious of MH and had been reported to the North American MH Registry using a clinical grading scale to evaluate the likelihood of truly being MH. Only two cases (0.7%) were regarded as ”very likely“ or ”almost certain“ being MH, after SCh had been applied to the patients but not a volatile anaesthetic . Recentlentified .2, enabling a differentiation between MH susceptible and normal pigs [2, PaCO2 and lactate as well as muscular and femoral PCO2 in susceptible pigs, are likely to be signs of a developing MH. Even if the alterations after SCh were less distinctive compared to the halothane-induced effects, our results documented a severe metabolic stimulation in the susceptible animals.In animals, intramuscular injection of halothane resulted in a local hypermetabolic response with locally limited rise of lactate and PCOmal pigs , while lmal pigs . Furthermal pigs ,14, Iaizmal pigs ,16. Howemal pigs . In contPossible explanations for SCh-induced MH may be based on the pharmacological characteristics of this agent. After intravenous injection, SCh activates the nicotinergic acetylcholine receptor, which acts as an unspecific cation channel provoking a local depolarization of the cell membrane. The transient depolarization of voltage-gated receptors and an influx of extracellular calcium via the nicotinergic acetylcholine receptors followed by an increase of intracellular calcium levels may trigger MH in predisposed individuals . In thisIn the current study, simultaneous application of halothane and succinylcholine potentiated the induced hemodynamic and metabolic effects compared to the changes following administration of halothane or SCh alone in MHS swine. Interestingly, a significant rise of body temperature only occurred after administration of a combination of both pharmacological agents, reflecting the severe metabolic breakdown. Our findings suggest an additive effect of SCh as postulated by previous investigations in which the onset of an MH episode was significantly enhanced and the increase of serum creatine kinase was more pronounced after a combination of SCh and volatile anaesthetics ,19,20.2 levels compared to PaCO2 and end-tidal PCO2 in MHS swine might be caused by a significant muscular venous outflow of carbon dioxide. Hence, blood gas analysis of venous blood might be as helpful as an arterial blood gas in clinical practice for real time detection of a developing MH episode.Hypermetabolism during a fulminant MH episode is typically associated with an excessive increase of end-tidal and arterial carbon dioxide concentrations ,6. The mIn conclusion, the findings of our investigation clearly demonstrated, that both SCh and halothane alone had the ability to induce an MH in susceptible swine. Furthermore, the observed hemodynamic and metabolic effects were excessively potentiated by combination of both pharmacological agents. Based on these results, SCh acts independent as well as supportive to provoke an MH episode in predisposed individuals. Hence, even if convincing reports of fulminant MH in humans induced by SCh alone are lacking, the obtained findings of our study proved the MH eliciting potency of SCh. However, the underlying mode of action of SCh to cause an MH episode still remains unclear and needs to be evaluated in further investigations.2: Arterial carbon dioxide pressure; PaO2: Arterial oxygen partial pressure; PCO2: End-tidal carbon dioxide partial pressure; SaO2: Peripheral oxygen saturation; SCh: Succinylcholine.BE: Base excess; IVCT: In vitro contracture test; MAP: Mean arterial blood pressure; MH: Malignant hyperthermia; MHN: Malignant hyperthermia non-susceptible; MHS: Malignant hyperthermia susceptible; PaCOAll authors declare that they have no competing interests.FS conceived the study, accompanied the data acquisition, collected the data and drafted the manuscript. SJ analyzed the data and helped writing the manuscript. SM analyzed the data. TM collected the data. NR participated in the design of the study. MA participated in the design of the study, collected and analyzed the data. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2253/14/14/prepub


Prevention of mother to child transmission of HIV (PMTCT) has been scaled, to more than 90% of health facilities in Tanzania. Disclosure of HIV results to partners and their participation is encouraged in the program. This study aimed to determine the prevalence, patterns and predictors of HIV sero-status disclosure to partners among HIV positive pregnant women in Morogoro municipality, Tanzania.A cross sectional study was conducted in March to May 2010 among HIV-positive pregnant women who were attending for routine antenatal care in primary health care facilities of the municipality and had been tested for HIV at least one month prior to the study. Questionnaires were used to collect information on possible predictors of HIV disclosure to partners.A total of 250 HIV-positive pregnant women were enrolled. Forty one percent (102) had disclosed their HIV sero-status to their partners. HIV-disclosure to partners was more likely among pregnant women who were < 25 years old , who knew their HIV status before the current pregnancy , and discussed with their partner before testing . Dependency on the partner for food/rent/school fees, led to lower odds of disclosure to partners . Nine out of ten women reported to have been counseled on importance of disclosure and partner participation.Six in ten HIV positive pregnant women in this setting had not disclosed their results of the HIV test to their partners. Empowering pregnant women to have an individualized HIV-disclosure plan, strengthening of the HIV provider initiated counseling and testing and addressing economic development, may be some of the strategies in improving HIV disclosure and partner involvement in this setting. HIV/AIDS and mother-to-child transmission of HIV (MTCT) are still major public health problems in sub Saharan Africa (SSA). SSA has an estimated 68% of the 33 million people living with HIV (PLWHIV) globally, nearly 90% of pregnant women living with HIV and more than 90% of HIV infections among children < 15 years old, globally [Prevention of mother-to-child transmission of HIV (PMTCT) programs are now available in many low and middle income countries . DisclosHIV testing of pregnant women should not only be the entry point to PMTCT of HIV, but also considered as an opportunity for prevention of sexual transmission of HIV. Studies have shown that, a substantial proportion of HIV infected individuals are in stable partnership with HIV uninfected partners in SSA. The prevalence of HIV-serodiscordance ranges from 8 -14% in eastern Africa and 14-27% in southern Africa ,18. A laThe prevalence of HIV sero-status disclosure to sexual partners has shown to differ between populations. Prevalence of disclosure among women attending VCT ranged between 69% - 86% ,11,23, 8In Tanzania the national PMTCT programme was introduced in 2004, and by the end of 2011, 91% of the health facilities with reproductive and child health (RCH) services were offering PMTCT service . While sThis cross sectional study was conducted between March and May 2010 among HIV positive pregnant women in Morogoro Municipality, Tanzania. Morogoro Municipality is one of the six districts of Morogoro region situated in south eastern Tanzania, about 190 kilometers west of Dar es Salaam. The district has an approximate population of 294,467, with the main economic activities for about 90% of the population being small scale farming, keeping livestock, small scale trade and working in the textile industry .Administratively, the district has one division, 19 wards and 274 hamlets. At the time of the study, the municipality had no district hospital, but it had four government health centres and 12 dispensaries. All of the 16 government health facilities offered RCH services including routine antenatal care, child growth monitoring, vaccination, family planning, HIV counseling and testing for pregnant women and offering antiretroviral prophylaxis and other necessary care for HIV positive women and their infants.Attendance for RCH services is high; 98% of women attend for antenatal care, 78% of children have been fully vaccinated and 61% deliver with skilled birth attendants, compared to the national level of 96%, 75% and 50% respectively . The HIVSimple random sampling was used to select six government health facilities out of 16 to participate in the study. Three health centres and three dispensaries participated. All the HIV positive pregnant women who were attending the antenatal clinics for routine care during the study period were eligible to participate. We limited the inclusion to those HIV positive pregnant women who had undergone counselling and received their HIV test results at least one month prior to the study.All the HIV positive women meeting inclusion criteria were invited to participate. After obtaining informed consent, two trained researchers administered a questionnaire during face to face interviews .Descriptive statistics was used to summarize the data. Bivariate analysis was used to examine associations between dependent variable (HIV disclosure to partner) and explanatory variables by using Ethical clearance was obtained from the Ethical Committee of Kilimanjaro Christian Medical University College, Tumaini University. Permission to conduct the research was also obtained from the Medical Officer in charge of Morogoro Municipality and the heads of the respective clinics where the study was conducted. Individual written informed consent was obtained from every HIV positive pregnant woman who agreed to participate in the study.A total of 250 HIV positive pregnant women out of 265 participated in the study (response rate 94%). Their age ranged from 17 to 41 years with a mean (±SD) of 27 ±5.3) years. A majority of the study participants were married/cohabiting (92.0%), had primary education (69.0%), were unemployed (78.8%), dependent on partner for rent/food or school fees (83%), had ≤ 2 children (79%), with age of the last born being > 2 years for 98% of the women. In Tables  years. AThe majority of the women (80.8%) learned of their HIV status during their current pregnancy and the time lapse from knowing their status to interview ranged from 1 to 84 months, (median of 5 months). Few women 15% (37) discussed with their partners about HIV testing before taking the test. Of all 250 women, 16% (39) of the pregnant women’s partners came to the clinics for HIV counseling and testing and 23% (58) knew their partners HIV status.All women reported to have received counseling on infant feeding options and on availability of antiretroviral drugs (ARVs) to reduce MTCT of HIV. Further, 9 out of 10 women reported to have received counseling on importance of using condoms, on importance of partner disclosure and on bringing partner for HIV counseling and testing, see Table The proportion of women who had disclosed their HIV serostatus to their partners/spouses was 41% (102/250). Of the 102 respondents who disclosed their sero-status to partners, 80% did so within 7 days after getting the results, while the rest revealed their sero-status sometime later.In total, 60% 150/250) of the pregnant women had disclosed their HIV status to either partner or other family member at the time of interview, see Figure 50/250 ofAmong 148 pregnant women who had not disclosed, 43% (64) planned to disclose to their partners in the future, while 84 women did not plan to disclose at all, to their sexual partners.Tables Discussion with partner before testing (OR =13.0), ever used condoms (OR =2.2), and knowledge of woman’s HIV status before current pregnancy (OR = 4.9) positively influenced HIV disclosure to the partner. Awareness of partners HIV status and was associated with HIV disclosure; 98% of pregnant women who knew their partners HIV status disclosed compared to 23% who didn’t, p < 0.001.Perception of benefit of disclosure and counselors advice also influenced disclosure. Women who perceived there is benefit were 3 fold more likely to disclose compared to those who didn’t perceive there is benefit OR 3.11 (95% CI 1.37-7.09). Also women who perceived counselor did not played a role in disclosure were less likely to inform their partners about their HIV status compared to those who appreciated counselors role OR 0.01 (95% CI 0.00 – 0.02).Religion, occupation of the women or their partner, partners age, parity, knowledge of PMTCT transmission or prevention were examined but were not associated with HIV sero-status disclosure.In logistic regression analysis Table , women hThis study shows that a relatively low proportion (41%) of mostly married/cohabiting HIV positive pregnant women attending antenatal care in Morogoro Municipality had disclosed their HIV sero-status to their partners. This prevalence of HIV sero-status disclosure is within the range of 17% - 46% reported among pregnant women in Tanzania, Uganda, Kenya, Burkina Faso, and Ivory Coast ,19,21,25There is improvement of HIV disclosure to partners among pregnant women in Tanzania from 17 -22% in 2001 to 41% in 2010 in the current study ,25. HoweFindings from this study showed that there is an association between financial dependency and HIV sero-status disclosure to partners. Participants who depended on their partners for food, house rent, and school fees were less likely to disclose their HIV-serostatus to their partner/spouse, than others. Other markers of low economic status such as low income and lower educational level of the woman or her partner also affected disclosure negatively in bivariate analysis. Previous studies in Tanzania and South Africa showed that women with low social economic status or financial dependence on their partner tend to disclose to relatives or others rather than their own partner ,26. FearWomen who had discussed HIV testing with their partners before undertaking the HIV test were seven times more likely to disclose to their partner than those who didn’t ,26,32. ITime of HIV diagnosis significantly influenced HIV disclosure to partners. Women who knew their HIV status for more than 12 months or knew their status before pregnancy disclosed their HIV status more than others. It might be due to the fact that they had enough time to think and accept their status, therefore finding it easier to disclose, than those who were recently diagnosed and were still in shock and struggling with the HIV diagnosis . DisclosOur results also point to missed opportunities by the health system of diagnosing women of reproductive age earlier for HIV. Most of the participants were multiparous 82% (202/250), and among them 98% had children ≥ to 2 years, . These women had multiple contacts with health facilities when they brought their children for vaccination, child growth monitoring or when they attended for family planning services. The need to integrate or strengthen PITC for HIV at reproductive and child health (RCH) service delivery points other than antenatal cannot be over emphasized, given that multiparous women in this study were significantly less likely to disclose compared to nulliparous women.This study had some limitations. Disclosure of HIV status is a sensitive topic among HIV positive women ,11,26. TIn conclusion, disclosure of HIV sero-status has been identified as an important strategy to reduce vertical and sexual HIV transmission in Tanzania ,5,29. WhIt is recommended that counselors should be encouraged to help women to develop an individualized disclosure plan especially targeting older women ≥ 25 years, multipara women and those who didn’t discuss with partners before testing. Strengthening and integration of PITC into other RCH services is recommended. Couple counseling and broader measures addressing women’s economic empowerment should be long term goals at improving HIV disclosure and partner involvement.The authors declare that they have no competing interests.ESK designed the study, collected the data, analysis, interpretation and drafted the manuscript. DJD advised on the design, participated in data analysis, interpretation and critical review of the manuscript. SEM provided advise on the design, data collection, interpretation and critical review of the manuscript. All the authors read and approved the final draft of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/13/433/prepubQuestionnaires.Click here for file


Analysis of the metaproteome of microbial communities is important to provide an insight of community physiology and pathogenicity. This study evaluated the metaproteome of endodontic infections associated with acute apical abscesses and asymptomatic apical periodontitis lesions. Proteins persisting or expressed after root canal treatment were also evaluated. Finally, human proteins associated with these infections were identified. Samples were taken from root canals of teeth with asymptomatic apical periodontitis before and after chemomechanical treatment using either NaOCl or chlorhexidine as the irrigant. Samples from abscesses were taken by aspiration of the purulent exudate. Clinical samples were processed for analysis of the exoproteome by using two complementary mass spectrometry platforms: nanoflow liquid chromatography coupled with linear ion trap quadrupole Velos Orbitrap and liquid chromatography-quadrupole time-of-flight. A total of 308 proteins of microbial origin were identified. The number of proteins in abscesses was higher than in asymptomatic cases. In canals irrigated with chlorhexidine, the number of identified proteins decreased substantially, while in the NaOCl group the number of proteins increased. The large majority of microbial proteins found in endodontic samples were related to metabolic and housekeeping processes, including protein synthesis, energy metabolism and DNA processes. Moreover, several other proteins related to pathogenicity and resistance/survival were found, including proteins involved with adhesion, biofilm formation and antibiotic resistance, stress proteins, exotoxins, invasins, proteases and endopeptidases (mostly in abscesses), and an archaeal protein linked to methane production. The majority of human proteins detected were related to cellular processes and metabolism, as well as immune defense. Interrogation of the metaproteome of endodontic microbial communities provides information on the physiology and pathogenicity of the community at the time of sampling. There is a growing need for expanded and more curated protein databases that permit more accurate identifications of proteins in metaproteomic studies. Culture-independent molecular microbiology methods have refined and redefined the knowledge of endodontic infections, revealing a diversity of species much broader than previously anticipated by culture Proteomics technologies have emerged as a large-scale analysis of differentially expressed proteins, allowing a better understanding of the overall physiologic profile of cells and tissues in a given condition m/z) ratio of the peptides. Afterwards, the peptide-sequencing data are searched against protein databases using specific tools A combination of liquid chromatography (LC) with mass spectrometry (MS) has become a powerful approach for the identification of proteins occurring in complex mixtures. In methods based on LC, hundreds of proteins or peptides are separated by chromatographic columns, detected, identified and quantified by mass spectrometry in a single operation Thus far, the only metaproteomic analysis of endodontic infections was performed by Nandakumar et al. This study was carried out in accordance with the guidelines of, and after approval by, the Ethics Committee at Estácio de Sá University, Rio de Janeiro, Brazil, and written informed consent was obtained from the patients.Samples were taken from patients who had been referred for root canal treatment or emergency treatment to the Department of Endodontics, Estácio de Sa University. Only teeth from adult patients (ages ranging from 20 to 39 years) with carious lesions, necrotic pulps, and radiographic evidence of apical periodontitis were included in this study. Samples were obtained from the root canals of 12 teeth with asymptomatic apical periodontitis and from aspiration of pus from two cases of acute apical abscess, which showed localized swelling, fever, lymphadenopathy, and malaise. No apparent communication from the abscess to the oral cavity or the skin surface was observed. Selected teeth showed no significant gingival recession and were free of periodontal pockets deeper than 4 mm.Samples from the root canals of teeth with asymptomatic apical periodontitis were taken as follows. After the tooth crown was cleansed with pumice, a rubber dam was placed and the tooth and the surrounding field were decontaminated by a protocol using 3% hydrogen peroxide followed by 2.5% sodium hypochlorite (NaOCl) solution. Complete access preparations were made using sterile burs without water spray. The operative field, including the pulp chamber, was again swabbed with 2.5% NaOCl, which was then inactivated with sterile 5% sodium thiosulfate. If the root canal was dry, a small amount of 10 mM Tris–HCl (pH 8.0) was placed in the canal. A K-type file no. 15 was introduced up to approximately 1 mm short of the root apex, based on radiographs, and used to gently file the canal walls. Afterwards, the fluid in the canal was aspirated using a sterile disposable syringe and transferred to a cryotube containing protease inhibitor phenylmethanesulfonylfluoride (PMSF) and immediately frozen at −80°C. This root canal sample was called S1. Samples from acute apical abscesses were taken by aspiration of the purulent exudate from the swollen mucosa over each abscess. The overlying mucosa was disinfected with 2% chlorhexidine (CHX), and a sterile syringe was used to aspirate pus, which was immediately injected into cryotubes containing PMSF and frozen.Root canal samples from teeth with asymptomatic apical periodontitis were also taken after chemomechanical procedures. Canals were instrumented at the same appointment in all cases by using BioRaCe instruments  with the working length (WL) established 1 mm short of the radiographic apex. Master apical files ranged from BR5 (40/.04) to BR7 (60/.04), depending on both the root anatomy and the initial diameter of the root canal. Patency of the apical foramen was confirmed with a K-type file no. 20 throughout the procedures. Irrigation was performed with either 2.5% NaOCl (6 teeth) or 2% CHX (6 teeth), using disposable syringes and NaviTip needles  inserted up to 4 mm short of the WL. Post-instrumentation (S2) samples were taken from the root canals as described for S1 samples.TTA CCG CGG CTG CTG - 3′). Positive and negative controls were included in each batch of samples analyzed. Positive controls consisted of DNA extracted from Enterococcus faecalis (ATCC 29212). Negative controls consisted of sterile ultrapure water instead of sample. All reactions were run in triplicate. PCR reactions were performed in 50 µL of reaction mixture containing 1 µM of each primer, 5 µL of 10× PCR buffer , 3.8 mM MgCl2, 2.5 U of Taq DNA polymerase (Fermentas) and 0.2 mM of each deoxyribonucleoside triphosphate . PCR amplifications were performed in a DNA thermocycler . Cycling conditions consisted of initial denaturation step at 95°C/2 min, followed by 36 cycles at 95°C/30 s, 55°C/1 min, and 72°C/1 min, and final extension at 72°C/10 min. PCR products were subjected to electrophoresis in a 1.5% agarose gel–Tris-borate-EDTA buffer. The gel was stained with GelRed  and visualized under ultraviolet illumination.Ten microliters from clinical samples were subjected to DNA extraction by using the QIAamp DNA Mini Kit , following the protocol recommended by the manufacturer. Presence/absence of detectable bacteria in clinical samples was determined by using end-point PCR with universal 16S rRNA gene primers 8f (5′- AGA GTT TGA TYM TGG C - 3′) and 519r  connected to nanoflow LC (nLC-MS/MS) by an EASY-nLC system  through a Proxeon nanoelectrospray ion source. Peptides were separated by a 2%–90% acetonitrile (ACN) gradient in 0.1% formic acid using an analytical column PicoFrit Column , at a flow of 300 nL/min over 45 min. The nanoelectrospray voltage was set to 1.7 kV and the source temperature was 275°C. All instrument methods for the LTQ-Orbitrap Velos were set up in the data-dependent acquisition mode. The full scan MS spectra  were acquired in the Orbitrap analyzer after accumulation to a target value of 1e6. Resolution in the Orbitrap was set to r = 60,000 and the 20 most intense peptide ions with charge states ≥2 were sequentially isolated to a target value of 5,000 and fragmented in the linear ion trap by low-energy collision induced dissociation (CID) as dissociation or fragmentation method, normalized collision energy of 35%. The signal threshold for triggering an MS/MS event was set to 500 counts. Dynamic exclusion was enabled with an exclusion size list of 500, exclusion duration of 60 s, and repeat count of 1. An activation q = 0.25 and activation time of 10 ms were used. Samples were analyzed in duplicate.http://www.uniprot.org) in September 2012, with carbamidomethylation as fixed modification, oxidation of methionine as variable modification, one trypsin missed cleavage and a tolerance of 10 ppm for precursor and 1 Da for fragment ions. All datasets were processed using ScaffoldQ+v.3.3.1 software with false discovery rate less that 1% as cut-off. Gene Ontology annotation for functions category  were obtained using UniProt tools and database.Peak lists (msf) were generated from the raw data files using Proteome Discoverer version 1.3 (Thermo Fisher Scientific) with Sequest search engine and searched against Human  and Bacterial  protein database downloaded from UniProt . The system was equipped with an HPLC-Chip (Agilent Technologies) that incorporated a 360 nL enrichment column and a 150 mm ×75 µm reverse phase column was packed with Polaris-C18, 3 µm particles. Three analytical replicates were analyzed by mass spectrometry. For each mass spectrometry experiment, peptides were loaded onto the enrichment column with solvent A (water with 0.1% formic acid). A two-step gradient generated at a flow rate 0.3 µL/min was used for peptide elution. This included a linear gradient from 3% solvent B (acetonitrile with 0.1% formic acid) to 40% B over 40 min followed by a sharp increase to 90% B within 5 min. The total running time, including column reconditioning, was 65 min. The column effluent in all cases was directly analyzed by the 6530 QTOF mass spectrometer that was interfaced with an HPLC-Chip Cube nanospray source. The latter was operated at a capillary voltage of 1950 V with a capillary current of 0.085 µA in extended dynamic range (2 GHz) mode. The MS data were acquired in the positive ionization mode using Agilent MassHunter Workstation QTOF B.04.00. During the course of data acquisition, the fragmentor voltage, skimmer voltage, and octopole RF were set to 150, 65, and 750 V, respectively. Auto-MS/MS was performed using scan speed varied based on precursor abundance option with a maximum cycle time of 6.8 s. In each cycle, MS spectra were acquired at 8 Hz (eight spectra/s) (m/z 295–1700), and the twenty most abundant ions  exceeding 1000 counts were selected for MS/MS (m/z 50–1700). A medium isolation (4 m/z) window was used for precursor isolation. A collision energy table  in 6530 QTOF. Reference mass correction was activated using a reference mass of 1221.9906. Precursors were set in an exclusion list for 0.15 min after one MS/MS spectra.m/z were merged within a time frame of ±60 s. The UniProt microorganism and human database was searched for tryptic/non-tryptic peptides with a mass tolerance of 10 ppm for the precursor ions and a tolerance of 50 ppm for the fragment ions. Two missed cleavages, fixed Modification (carbamidomethylation) and variable modifications [oxidized methionine (M)] were allowed. Spectrum Mill autovalidation was performed at peptide and protein level (1% FDR).LC-QTOF data was searched against the UniProt micro-organism and human database, using the Agilent Spectrum Mill Server software (Rev B.04.00.127). Data were extracted and peak lists were created with the Spectrum Mill Data Extractor program with the following attribute: scans with the same precursor ±0.03 Identified bacterial proteins were categorized according to biological function as follows: transcription and translation, cell division/peptidoglycan synthesis, chemotaxis, DNA process, energy metabolism, fatty acid metabolism, methanogenesis, membrane, nucleotide metabolism, antibiotic resistance, adhesion, pathogenesis/virulence, proteolysis, stress response, transport, vitamin biosynthesis, and other/unknown. Human proteins were classified as follows: cellular process and metabolism, immune system, circulatory system, extracellular connective matrix, and other/unknown. Keratins and other presumed contaminants were removed from the entire dataset.PCR analysis of the individual clinical samples using universal 16S rRNA gene-based primers revealed the presence of bacteria in all S1 and abscess samples. Of the S2 samples, 4/6 samples from the NaOCl group and 5/6 samples of the CHX group showed positive results for bacteria.Analyses of the metaproteomic data generated by the two mass spectrometers revealed a total of 308 proteins of microbial origin . A largeIn abscess samples, proteins involved with transcription and translation processes were more frequent (30 proteins), followed by proteins associated with energy metabolism (21 proteins) and DNA processes (18 proteins). In rootEight proteins with proteolytic activity were found and most of them (six) occurred in abscesses. Proteins participating in bacterial adhesion were detected in one abscess sample, as well as in S1 and S2 samples from the NaOCl group. Proteins involved in production of biofilm matrix were found in one pool of S1 samples. Two CRISPR -associated proteins were found in one pool of S1 samples and S2 from the NaOCl group. An archaeal protein linked to production of methane was detected in one pool of S1 samples. Analyses of human proteins using the two mass spectrometers revealed 139 proteins, which were classified as follows: cellular process and metabolism (87 proteins), circulatory system (9 proteins), extracellular connective matrix (2 proteins), immune system (24 proteins), and other/unknown functions (17 proteins). Defensins 1 and 3 were identified in one abscess sample, one pool of S1 samples and the pool of S2 samples from the NaOCl group. Proteins involved with the host defense to infection are shown in This study evaluated the metaproteome associated with endodontic infections by using two mass spectrometry platforms. The large majority of proteins found were related to metabolic and housekeeping processes, including protein synthesis, energy metabolism and DNA processes. This is indicative of a living active microbial community. Moreover, several other proteins of interest were detected, including some related to pathogenicity and resistance/survival. Because these proteins may assume special relevance in terms of pathogenesis of apical periodontitis and resistance to host defenses and treatment, they deserve a more detailed discussion as follows.Biofilm formation is an important virulence trait of many bacterial pathogens Eight proteins with proteolytic activity were found in this study, most of them associated with abscesses. They included a collagenase, a metalloprotease, a serine protease, an extracellular protease, and endopetidases. These enzymes may play an important role in several ecological and pathogenic effects, including tissue invasion, acquisition of nutrients from proteins, destruction of the connective tissue matrix, and inactivation of host defense molecules Streptopain (SPE B), a protein of streptococcal origin, was found in one pool of S1 samples. This is a cysteine protease-like exotoxin with several biological effects, including cleavage of fibronectin, vitronectin, and interleukin 1-β precursor and participation in the apoptosis of monocytes and epithelial cells Several stress proteins were detected in the samples tested. Cells respond to environmental stress by inducing or accelerating the synthesis of specific proteins known as stress proteins, including heat-shock proteins (HSPs), which act as molecular chaperones in the assembly and folding of proteins, as well as proteases when damaged proteins need to be degraded. In addition to being found intracellularly, HSPs can also be located on the cell surface and on outer membrane vesicles released by bacteria Others stress response proteins detected in this study include alkyl hydroperoxide reductase, whose expression has been shown to be up-regulated in some anaerobes upon oxidative stress Catalase and quinone oxidoreductase are involved in the response to oxidative stress A toxin component of the putative toxin-antitoxin system was detected in an abscess sample. This system may mediate the general stress response, and participate in the regulation of biofilm and persister cell formation, which is an important mechanism of reduced susceptibility of biofilms to antibiotics Proteins involved in DNA repair are also of interest because they may participate in the response to stress conditions induced by treatment or host defenses. Some of the DNA repair-related proteins detected in this study were A/G-specific adenine glycosylase, DNA ligase, ATP-dependent helicase, and UvrABC system proteins. Of the 12 proteins related to DNA repair, 8 were found in abscesses; in these cases, there may be a strong oxidative stress caused by phagocytes during the combat to infection Two enzymes that confer resistance to antibiotics were detected: beta-lactamase  and TetR (resistance to tetracyclines). Previous studies have detected the genes for these antibiotic resistance enzymes directly in root canals samples mcrA, which encodes for the methyl coenzyme M reductase Another noteworthy finding was the identification of the enzyme methyl coenzyme M reductase of archaeal origin. Methanogenic archaea have been detected in endodontic infections by DNA-based molecular methods, including by an assay directed to CRISPR consists of identical repeated DNA sequences interspaced by highly variable sequences (spacers). CRISPR-associated (cas) genes encode conserved proteins that together with CRISPRs compose the CRISPR/Cas system, which is present in many prokaryotes and confer protection against invasion by phages, plasmids, and transposons Human proteins were identified in all samples, being more frequent in abscess samples and in S1 samples from asymptomatic teeth. The fact that most proteins identified are intracellular and are involved in cellular and metabolic processes can be possibly explained by rupture of cells during the inflammatory process in response to infection, especially in abscesses. Another possible source of intracellular proteins is the remnants of necrotic pulp tissue in the canal. However, it must be recognized that cellular rupture may also have occurred during sample taking or processing steps. In addition, 24 proteins related to the innate or adaptive immune system were found. They include defensins 1 and 3 and myeloperoxidases, which are produced by polymorphonuclear neutrophils. These proteins are not only involved with defense against microorganisms but also in tissue destruction Metaproteomic analyses revealed a larger number of proteins in the abscess samples when compared to samples from asymptomatic infection, in spite of the fact that samples from asymptomatic teeth came from a larger number of cases. Studies have shown that the number of bacterial cells and species in abscesses are greater than in asymptomatic cases Analysis of the total number of proteins detected after treatment revealed a decrease in the CHX group but an increase in the NaOCl group. The purpose of analyzing post-instrumentation samples was two-fold: first, to evaluate the effects of treatment on the metaproteome; second, to evaluate if the proteins expressed after treatment procedures would differ from those present before treatment, considering the possibility that microorganisms resisting to treatment may produce a different pattern of proteins. The reduction in the CHX group was completely expected, but the increased in NaOCl group may be related to the tissue dissolving ability of this substance and its high destructive effects on cells, releasing cytoplasmic proteins to the environment. This is coherent with the types of proteins found in S2 samples from the NaOCl group. In the group of CHX, one stress response protein was found, and this may be related to a response to treatment. However, a more distinct pattern was not evident. This may have been due to one of the following reasons: limitation of the sampling approach; few bacterial cells remaining after treatment; and the short time elapsed between treatment and sampling, not allowing sufficient time for bacteria to change their pattern of protein expression in order to adapt themselves to the altered environment. If the latter is true, further studies should be performed to analyze the metaproteome associated with bacterial persistence after treatment, and for this, researchers should wait a little longer to collect samples after intracanal procedures.Actually, in the present study, efforts were expended towards evaluation of the exoproteome. However, many cytoplasmic proteins were detected. This may be due to cells that recently died and released their protein content in the environment , which are still considered as part of the exoproteome if these proteins are stable and remain in abundance Several proteins identified in this study were not linked to endodontic bacteria, which may be related to databases with incomplete information for oral bacteria The number of proteins identified may have also been affected by the principle of parsimony adopted by the softwares used in this study, which reduce the occurrence of redundant sequences In contrast to bacterial isolates cultivated in the laboratory, human clinical samples, especially from a small environment like the root canal, provide a reduced amount of biomass available for proteomic analysis. Therefore, for root canal samples to be properly analyzed they had to be pooled. In addition, many proteins were found only by one of the methods used. This may be related to the different aliquots used in each identification, which may influence detection of proteins found in low abundance. The peptide mixture generated by the shotgun proteomic strategy is so complex to permit that the mass spectrometer acquires the mass spectra of all peptides in a single LC-MS/MS run. Consequently, these experiments must be frequently repeated so as to increase the number of peptides obtained by mass spectra Interrogation of the metaproteome of endodontic microbial communities provides information on the physiology and pathogenicity of the community at the time of sampling. The present study qualitatively described the proteins of microbial and human origin in association with endodontic infections. There is a growing need for expanded and more curated protein databases that permit more accurate identifications of proteins in metaproteomic studies.The authors are grateful to Vadi Bhat, from Agilent Technologies, for the technical support with the 6530 QTOF mass spectrometer.Table S1Microbial proteins identified in the metaproteome analysis of symptomatic and asymptomatic endodontic infections and after treatment(XLS)Click here for additional data file.Table S2Human proteins identified in the metaproteome analysis of symptomatic and asymptomatic endodontic infections and after treatment(XLS)Click here for additional data file.


Taraxacum) comprise a group of sexual diploids and apomictic polyploids with a complicated reticular evolution. Apomixis  in this genus is considered to be obligate, and therefore represent a good model for studying the role of asexual reproduction in microevolutionary processes of apomictic genera. In our study, a total of 187 apomictic individuals composing a set of nine microspecies (sampled across wide geographic area in Europe) were genotyped for six microsatellite loci and for 162 amplified fragment length polymorphism (AFLP) markers. Our results indicated that significant genetic similarity existed within accessions with low numbers of genotypes. Genotypic variability was high among accessions but low within accessions. Clustering methods discriminated individuals into nine groups corresponding to their phenotypes. Furthermore, two groups of apomictic genotypes were observed, which suggests that they had different asexual histories. A matrix compatibility test suggests that most of the variability within accession groups was mutational in origin. However, the presence of recombination was also detected. The accumulation of mutations in asexual clones leads to the establishment of a network of clone mates. However, this study suggests that the clones primarily originated from the hybridisation between sexual and apomicts.Dandelions (genus  Taraxacum), the widespread distribution of apomictic clones suggests that they are temporarily ecologically successful Paradox of SexAsexual reproduction through seeds  occurs in less than 1% of flowering plants The lack of recombination is expected to direct apomicts towards their extinction Taraxacum, Hieracium, Rubus, and Poa, with significant geographic parthenogenesis in their distribution Apomicts have significant advantages over sexuals in colonising new areas Taraxacum Wigg.  consists of perennial herbs that are widely distributed throughout the world (with exception of Antarctica). The putative centre of origin is in Central Asia in a region that includes the Himalayas Taraxacum is obligate meiotic diplospory, which is the type that is most similar to sexual reproduction among apomixis systems DIPLOSPOROUS (DIP) and PARTHENOGENESIS (PAR) The genus Taraxacum form polyploid series; apomicts are mostly triploids or tetraploids, whereas sexuals are mostly confined to diploids Taraxacum species are classified into morphological groups (sections) that contain one or more sexual species and polyploid clumps of apomictic accessions . Apomicts are either of autopolyploid origin or are the result of hybridisation Species of Taraxacum populations comprise individuals with extended genotypic variability; such genotypes of these populations can be widely distributed or can exist as local clones et al.Naevosa. He stressed that individuals from a single clone with sufficiently long asexual histories may differ genetically due to mutation accumulation.Fully asexual or mixed sexual apomictic Taraxacum officinale agg. sampled in regions of sympatric occurrence of sexuals and apomicts, allowing us to study the mutation load and the formation of novel genotypes in greater detail. Morphology was the key criterion for accession assignment. In present study, we addressed following questions: 1) What is the pattern of genotypic variability within and differentiation among apomictic clones? 2) What is the source of intraclonal and interclonal variability? 3) What is the origin of apomictic clones? 4) What is the detection ability of molecular markers? To answer these questions, we used three types of molecular markers. Microsatellites are flexible tools for population studies  planies e.g. . The devTaraxacum officinale agg., i.e., T. sect. Taraxacum  T. amplum agg. – AMP group, each phenotyped accession denoted as amp1– amp6); the second one contains three morphologically divergent accessions . Term “accession” is used in sense of morphologically homogeneous phenotypic unit. All accessions were phenotyped according to the taxonomic microspecies concept and only confirmed phenotypes were included in this study; a complete list of the individuals studied (including taxonomic identification) is provided in We studied total of 187 individuals from two morphological series of apomictic accessions of OL).We sampled one individual per one locality across the wide geographic range of Central Europe . The platrnL-trnF region, four sexual diploid plants were added. Ploidy levels were confirmed by flow-cytometric analysis of relative DNA content using an inner diploid control.Apomictic reproduction was confirmed by either emasculation or Flow Cytometric Seed Screen (FCSS) Genomic DNA was extracted from voucher specimens or fresh leaves, following CTAB (Cetyl Trimethyl Ammonium Bromide) protocol of Doyle & Doyle The research did not involve any endangered or protected plant species, originating from restricted privately-owned or protected territories, so no specific permits were required for plant sampling.2 in final volume) and 0.42 U of GoTaq DNA Polymerase (Promega). Detailed information on PCR conditions  is available upon request.All 187 individuals were genotyped for six microsatellite loci: MSTA44B, MSTA53, MSTA78 EcoRI-AGC/MseI-CAAT, EcoRI-AAT/MseI-CAAC, and EcoRI-AGC/MseI-CGATG). AFLP analyses followed the protocol of Vos et al.et al.Ninety-six individuals including all nine apomictic accessions see  were anatrnL-trnF region was sequenced in four samples from each apomictic accession and in the diploid sexual. A PCR reaction was performed in a total volume of 25 μl with 10 ng of template DNA, 2 μM of e and f primers 2) and 1 U of Pfu DNA Polymerase (Fermentas). The reaction conditions were as follows: 95°C for 2 min; 30 cycles with 95°C for 1 min, 52°C for 1 min, and 72°C for 1 min; followed by 5 min at 72°C. The PCR products were sequenced using an Applied Biosystems 3730xL capillary sequencing system. Sequences were edited in BioEdit ega 5 The PCR products were separated by denaturising polyacrylamide gel electrophoresis (PAGE) and visualised by silver staining. The 30–330-bp AFLP® DNA ladder (Invitrogen) was used to size the microsatellite alleles. If only one allele was observed for a locus, then the individual genotype was considered to be an absolute homozygote; when two different alleles were observed, the third was coded as missing data. In the case of AFLP, the profiles were visually checked and coded as a binary matrix. To avoid the genotyping errors and to retain reproducibility of the analyses, several control levels were included during the entire study: blind samples, double samples and repetitions. This allowed estimation of the error rate, which was calculated as the difference between all markers and the markers used in the final matrix.rlequine 3.5 actax from package Pica 4.0 MIC – the matrix incompatibility count) between each pair of multilocus genotypes is computed. After removing of genotypes with the highest number of incompatibilities only fully compatible genotypes should be left in the matrix differing only due to mutations (MIC  = 0) Microsatellites data were analysed as microsatellite genotypes based on the number of repeats. They were also scored using a binary matrix, where the presence/absence of a fragment of a particular size was coded as 1/0. This approach was used to perform hierarchical AMOVA with AeDi software NG), overall number of alleles, the number of different alleles (NDA), the means allele size (MAS), the range of allele size, and gene diversity (e/GeG). To characterise polymorphism and among-population differentiation, locus and multilocus estimates of F- and R-statistics were calculated. The contribution of stepwise mutations (SMM) vs. nonstepwise mutations  to population differentiation was tested  From genotype data several locus and multilocus statistics were computed using SPAGNB) and the number of polymorphic bands (NPB) at the 5% level, number of private markers , number of diagnostic markers  were calculated in FamdP), Nei’s gene diversity (jH), number of different genotypes (NG) and genotype diversity (GD) were calculated using the R-script of AFLPdatBasic population statistic indices such as the mean number of bands , was done in SplitsTree 4  was performed in NTSYS-tructure 2.2 aps 3.2 tructure and Baps is in the treatment of K (number of clusters). Whereas Structure uses the Markov Chain Monte Carlo (MCMC) algorithm to cluster genetically similar individuals and to estimate the likelihood of the data for different numbers of groups (K), in Baps frequency of alleles and the number of genetically different groups are taken as random variables and the program estimates one optimal partitioning. Computation in Structure was set up for the recessive allele model and the admixture model with correlated allele frequencies. The K was set to 1–11 with 10 replicate runs for each K using the 1,000,000 MCMC iterations following the period of 100,000 burn-in iterations. The computation was carried out on the freely accessible Bioportal of the University of Oslo . The R-script Structure-sum-2009 SC), means of the posterior log probability [meanL(K)], and a quantity based on the second order rate of change of the likelihood function with respect to K (ΔK) . Most of genotypes were clonal or differ in only one or two alleles in a few repetitions. No variability was observed among all genotyped individuals of S, with only one genotype detected over all of the loci. For O, P, amp4, two genotypes were observed for only one locus, and all other loci were without genotype variation. The rest of the species show higher genotype variability within genotyped loci. Individuals of amp1 showed the highest genotype and allelic variability  to eG  = 0.89 (MSTA44B)  , Table 2RST and FST were similar (FST for description of variability for multilocus and majority of locus estimates (data not shown). Nevertheless, locus estimates for MSTA133 (P<0.05) and MSTA78 (P  = 0.059) within AMP+OSP and MSTA78 (P<0.01) within OSP suggested that some loci may undergo also stepwise mutations. As expected from the nature of the plants used in this study, the inbreeding coefficient FIS/RIS reached high negative values. In multilocus estimates, FIS/RIS  =  −0.5400/−0.5744 for the AMP+OSP group , the pattern of the variation remains unchanged with 22.9% of variation between AMP/OSP groups.No monomorphic alleles were observed for loci MSTA78 and MSTA53 across the analysed samples, which should be linked the amp1, amp2, amp3, amp5, amp6, S, OSP, and AMP. For others apomictic accessions, fewer than four required genotypes were present  was found, and in amp2, amp3, amp6, only one genotype cause matrix incompatibility; after its removal, MIC  = 0  in the amp1 accession, and among ten AFLP phenotypes, only seven also had different SSR genotypes. In amp3, ten AFLP phenotypes were detected, but only four also had different SSR genotypes. The difference occurred in one or two alleles in one repetition. Amp4 contained five AFLP phenotypes, and only one also had a different SSR genotype in one repetition.Three primer combinations produced a total of 162 unambiguously scorable markers, of which 129 were polymorphic. The error rate corresponds to 2%. An observation of clonality  showed that most of the clonal genotypes in ividuals . The higamp1 to the lowest value of only 1.9% for O. For each apomictic accession, private and diagnostic markers were detected, only for amp1 no diagnostic marker was observed  and amp3 (jH  = 0.053). The distribution of genotypic variability revealed that the majority of the variability, 86.6%, occurred among apomictic accessions, while only 13.4% occurred within accessions. The addition of one more hierarchical level  did not change the variability distribution, with 51.6% variability among apomictic accessions and 37.6% among groups  increased up to two and then flattened out. Additionally, ΔK showed a maximum value for K  = 2 (SC  = 1). Because a high similarity coefficient (SC  = 0.8) was also observed for K  = 3, this clustering was inspected. For the K  = 2, Structure identified clusters corresponded with the division of the AMP and OSP groups. For K  = 3, two different clustering outcomes were gained  . The dive gained .amp1, 1-amp6, 1-P) because of high background noise in the sequences. Only five haplotypes were observed within accessions (1b for amp2 and cp3 for P), while two appeared only once and were individual specific (cp1c and cp2 within amp1). The most common haplotype, cp1a, was shared among the rest of the accessions and diploids. The differences between each haplotype and the most common haplotype, cp1a, were as follows: substitution for cp1b, one deletion; for cp1c, nine substitutions and two deletions; and for cp3, the insertion of 9 bp, six substitutions, and one deletion.The length of the final alignment was 384 bp. Three samples were discarded from the alignment (1-cessions . Two of Apomictic dandelions (with diplospory as the prevailing reproduction mode) are widely distributed in temperate zones of the Northern hemisphere. These apomicts usually predominate in populations in cooler regions and the genus as a whole is successful in colonizing great areas. In this study, we tested whether morphologically uniform phenotypes  are genetically uniform or diverged. Our data showed that apomictic accessions are genetically highly homogeneous and that its low genotypic variability can be explained by the accumulation of mutations during their asexual history. Many of the observed genotypes were clonal and accession specific and therefore accessions could be considered to represent apomictic clones. However, the variability among accessions is much higher than within accession variability. It originates from the recombination events – sexual process between apomictic pollen donor-apomictic father and sexual mother. The pattern of variability and its distribution is in correspondence with the morphological and taxonomic differentiation of accessions into apomictic microspecies.Taraxacum apomicts is caused by mutation load within a single clone but that interclonal diversity is most likely of sexual origin. We found that all of the investigated apomictic accessions contained nearly identical genotypes or a low number of different genotypes  complex, where lower number of genotypes than AFLP phenotypes was detected by 13 SSR loci Ranunculus carpaticola had as many genotypes as individuals for two SSR loci Taraxacum albidumR. carpaticolaWe found a perfect correlation between genotype fingerprints and phenotypes. All of the observed genotypes were accession specific, with no genotype shared among them. A similar pattern of genotypic diversity was observed within both markers examined in this study. With AFLPs, we detected nearly as many AFLP phenotypes as observed individuals for the cessions . Despite−2–10−6; , the acc−2–10−6; , Table 4et al. The terminology used for clones varies among geneticists, plant systematists and molecular biologists. For taxonomists or ecologists a clone usually refers to morphologically identical offspring of vegetative (or apomictic origin), whereas strictly genetic view differs in considering each unique genotype or AFLP phenotype as distinct clone even they are morphologically identical. Our data support the hierarchy of apomictic clones discussed by van Dijk Genotypic variability in the morphological groups of apomictic dandelions that we studied had similar distributions for both types of markers. Analysis on several hierarchical levels showed that diversity is distributed mainly among accessions, whereas higher homogeneity was observed within accessions .OSP and AMP clearly form two separate pools of apomictic genotypes, which were confirmed by testing the relatedness of genotypes using several clustering methods  in population samples of sexuals from France Some microsatellite loci are highly conserved and are thus shared among individuals from distant regions. Microsatellites linked to OSP only two genotypes do not fit the expectation for genotypes differentiating by changes gained through clonal reproduction. While the results could suggest that genotypes within the OSP group differ purely by accumulation of mutations, the results from cpDNA revealed that P had a different origin from S and O  and a consequent radiative spreading of the clones. The possible scenario of the AMP group origin is the hybridisation between the sexual and apomictic preamp1 genotypes, in which an array of newly asexual genotypes arose and several lineages became fixed and further evolved into separate clones that share a common history. This scenario confirmed also the result of Matrix Compatibility analysis, in which nearly all genotypes are in congruence with recombinational/sexual difference of investigated accessions  hybridisation. The structure of their genotypic variability is tightly correlated with morphology. Correct genotyping is crucial requirement both in population biology and ecological studies Our data demonstrate that there is both significant genetic similarity and significant differences among putative apomictic clones that were identified by phenotype. Unlike in previous studies, in which clonality was detected in a population sample of not-phenotyped accessions, we focused on the genetic structure within phenotyped apomictic accessions themselves. Both asexual life history and sexual recombination have an impact on the genetic variability of apomicts of Table S1List of apomictic Taraxacum accessions used in this study with sampling details. Country abbreviations: CZ – Czechia; SK – Slovakia; collector abbreviations: BT – Bohumil Trávniček, RJV – Radim J. Vašut, LM – Ľuboš Majeský. The columns: F/H refers to plant material used for DNA extraction, F – fresh leaves, H – herbarium voucher, FB – flower buds; FCSS/EM refers to method used for examination of reproduction type, FCSS – Flow Cytometry Seed Screen, EM – emasculation, apo – apomictic seed formation; for T. linearisquameum FCM is showed where 2x  =  diploid sexual; SSR/AFLP/cpDNA shows which plants were used for SSR – microsatellite genotyping, AFLP – genotyping, cpDNA - sequencing of trnL-trnF region with observed haplotype + GeneBank accession number; b – double sample. Asterisk (*) indicates accessions recognized by taxonomists as validly described microspecies; absence of asterisk indicates distinct morphological groups but formally undescribed accessions (mentioned under “work names”).(DOC)Click here for additional data file.


Sedation after open heart surgery is important in preventing stress on the heart. The unique sedative features of propofol prompted us to evaluate its potential clinical role in the sedation of post-CABG patients.To compare propofol-based sedation to midazolam-based sedation after coronary artery bypass graft (CABG) surgery in the intensive care unit (ICU).Fifty patients who were admitted to the ICU after CABG surgery was randomized into two groups to receive sedation with either midazolam or propofol infusions; and additional analgesia was administered if required. Inclusion criteria were as follows: patients 40-60 years old, hemodynamic stability, ejection fraction (EF) more than 40%; exclusion criteria included patients who required intra-aortic balloon pump or inotropic drugs post-bypass. The same protocol of anesthetic medications was used in both groups. Depth of sedation was monitored using the Ramsay sedation score (RSS). Invasive mean arterial pressure (MAP) and heart rate (HR), arterial blood gas (ABG) and ventilatory parameters were monitored continuously after the start of study drug and until the patients were extubated.The depth of sedation was almost the same in the two groups  but the total dose of fentanyl in the midazolam group was significantly more than the propofol group (12.5 mg/hr vs 4 mg/hr) (P = 0.0039). No significant differences were found in MAP (P = 0.51) and HR (P = 0.41) between the groups. The mean extubation time in patients sedated with propofol was shorter than those sedated with midazolam  but the ICU discharge time was not shorter .Propofol provided a safe and acceptable sedation for post-CABG surgical patients, significantly reduced the requirement for analgesics, and allowed for more rapid tracheal extubation than midazolam but did not result in earlier ICU discharge. Sedation after open heart surgery is very important because most cardiac surgeons and anesthetists prefer CABG patients to awake slowly, to prevent any stress on the heart. Most patients require both sedation and analgesia to promote natural sleep, facilitate assisted ventilation, and modulate physiologic responses to stress (e.g. tachycardia and hypertension) -3. Pain To evaluate the potential clinical role of propofol in the sedation of post cardiac surgery patients and to compare the sedative properties, safety profiles, cardiovascular responses and ventilation and extubation characteristics of propofol with those of the commonly used i.v. sedative agent midazolam in the cardiac intensive care unit.In this randomized clinical trial, all the consecutive patients undergoing elective CABG in Shahid Rajaei Hospital, Tehran, Iran were enrolled in the study. After the study protocol was approved by the Ethics Committee of Shahid Rajaei Hospital, all of the 50 adult patients signed informed written consent to participate in this study. Fifty adult patients, who had undergone coronary artery bypass graft surgery, were postoperatively assigned to one of two treatment regimens for sedation, if it was expected that they would require a minimum of 8 h mechanical ventilation after the surgery. These patients were ASA physical status II, 40-60 years old and had ejection fraction (EF) ≥ 40% who were scheduled for elective coronary artery bypass graft surgery under general anesthesia. Exclusion criteria were patients with underlying and co-existing diseases , opium addiction and patients requiring post-bypass intra-aortic balloon pump or inotropic drugs. Protocol of anesthetic medications used in all of patients was the same and according to standard clinical practices. Before the induction of anesthesia, all patients were pre-medicated with 1 mg intramuscular lorazepam and 0.1 mg/kg morphine sulfate one hour before entering the operating room. Induction of anesthesia was performed under the monitoring of ECG, pulse oximetry and invasive arterial blood pressure with 0.2 mg/kg etomidate, 2.5 μg/kg sufentanil and 0.2 mg/kg cisatracurium and maintenance of anesthesia after the insertion of central venous line was achieved with continuous infusion of midazolam, sufentanil, and atracurium in both groups. On arrival in the ICU, patients were allocated randomly, using sealed envelopes provided by the supervisor of ICU to receive i.v. infusions of either midazolam  or propofol  while being mechanically ventilated, together with the short‐acting opioid fentanyl by continuous infusion, for analgesia if required. An initial loading dose infusion of midazolam or propofol was given to rapidly achieve a steady‐state plasma concentration. The loading dose infusion of midazolam was 0.05 mg/kg over 10 min followed by a maintenance infusion of 0.04- 0.1 mg/kg/h using a peripheral or central vein. Propofol was given undiluted (ropofol 1%) as an infusion of 1-3 mg/kg/hr, after a loading dose infusion of up to 1 mg/kg over 10 min. Fentanyl was infused at 1-5 µg/kg/hr after a bolus of 1 µg/kg if the patient was in pain. 2 every 30 minutes by 0.1, while a SpO2 ≥ 95% was maintained until an FIO2 ≤ 0.4 was reached. Extubation time was defined as the time from cessation of sedative infusion to extubation. The sedative and analgesic infusion was discontinued, in preparation for the extubation, when there was no evidence of bleeding and the patient was awake, cooperative and comfortable, cardiovascularly stable, normothermic, and with an acceptable blood gas on FIO2 ≤ 0.4, positive end-expiratory pressure ≤ 5 cm H2O, pressure support ≤ 10 cm H2O, tidal volume ≥ 5 mL/kg, and spontaneous respiratory rate < 20/min. The patient status was assessed every 30 minutes and recorded every 2 hours. Blood pressure and heart rate were monitored continuously but recorded every 30 minutes during the first hour after the start of study drug and hourly until study drug was stopped. SpO2 was monitored continuously. ABG, Na, K, Cl, Ca, Hgb, Hct, and lactate were checked every two hours until the extubation. Venous samples were taken for routine hematological  and biochemical  profiles immediately on arrival in the ICU, and then at 24 and 48 h; but the results are not shown in this report. Discharge from the ICU was performed if there were no signs of neurological (Ramsay sedation score 2), respiratory , hemodynamic  or surgical  impairment and if the pain score on the pain rating scale or visual analogue scale (VAS) was < 4.Since the most common sedation scale in the previous studies was the Ramsay scale, the degree of sedation was measured and recorded hourly using the Ramsay Sedation Score (RSS)  and patiThe statistical analysis of data was performed using the SPSS Version 15.0 statistical software  The intragroup differences of the circulatory variables recorded over the time were analyzed using the repeated measures analysis of variance. Differences in the mean values of circulatory variables and analgesic requirement were analyzed using independent samples t test. The categorical variables in the two groups were analyzed using Chi-square test or Fisher's exact test. Extubation time and ICU stay time variables were analyzed by K-S test for assessing the normal distribution; and if not distribute normally, Mann-Withney U test was used for the analysis. The hypothesis of this study was that there would be a clinically meaningful difference in the hemodynamic responses, extubation time, ICU stay and analgesic requirement between the groups receiving two different sedative drugs. The quantitative data were expressed as mean ± SD. A P value of ≤ 0.05 was considered statistically significant for all tests.There were no statistically significant differences between the two patient groups with respect to age, weight, gender and left ventricular ejection fraction . Mean duUntil recently, ICU care focused on correcting medical/surgical issues without worrying about the oversedation or prolonged ventilation time. This randomized trial study was designed to compare the propofol based sedation for post-CABG patients with the midazolam-based sedation regimens in the ICU. In our study, an equivalent depth of sedation between midazolam and propofol receiving ICU patients was achieved. These results are consistent with previous studies in patients admitted to ICU after a variety of major surgeries , 25-27. Inadequate sample size to evaluate some issues such as the length of ICU stay.Sedation and analgesia are essential components of care for many mechanically ventilated patients in the intensive care unit (ICU). This study and several other studies during recent years have shown propofol to be an effective and safe agent for use as post‐operative sedation in the ICU. Propofol and midazolam are commonly used for the care of post–coronary artery bypass graft (CABG) patients in the ICU, but compared with midazolam, propofol provides equal or better control in maintaining the sedation and more rapid recovery, more rapid extubation when sedation is terminated and less requirement for analgesic drugs for pain control. These properties have advantages for patients at risk for myocardial ischemia.