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Sample records for abuja targets identifying

  1. Towards achieving Abuja targets: identifying and addressing barriers to access and use of insecticides treated nets among the poorest populations in Kenya.

    Science.gov (United States)

    Chuma, Jane; Okungu, Vincent; Ntwiga, Janet; Molyneux, Catherine

    2010-03-16

    Ensuring that the poor and vulnerable population benefit from malaria control interventions remains a challenge for malaria endemic countries. Until recently, ownership and use of insecticides treated nets (ITNs) in most countries was low and inequitable, although coverage has increased in countries where free ITN distribution is integrated into mass vaccination campaigns. In Kenya, free ITNs were distributed to children aged below five years in 2006 through two mass campaigns. High and equitable coverage were reported after the campaigns in some districts, although national level coverage remained low, suggesting that understanding barriers to access remains important. This study was conducted to explore barriers to ownership and use of ITNs among the poorest populations before and after the mass campaigns, to identify strategies for improving coverage, and to make recommendations on how increased coverage levels can be sustained. The study was conducted in the poorest areas of four malaria endemic districts in Kenya. Multiple data collection methods were applied including: cross-sectional surveys (n = 708 households), 24 focus group discussions and semi-structured interviews with 70 ITN suppliers. Affordability was reported as a major barrier to access but non-financial barriers were also shown to be important determinants. On the demand side key barriers to access included: mismatch between the types of ITNs supplied through interventions and community preferences; perceptions and beliefs on illness causes; physical location of suppliers and; distrust in free delivery and in the distribution agencies. Key barriers on the supply side included: distance from manufacturers; limited acceptability of ITNs provided through interventions; crowding out of the commercial sector and the price. Infrastructure, information and communication played a central role in promoting or hindering access. Significant resources have been directed towards addressing affordability

  2. Towards achieving Abuja targets: identifying and addressing barriers to access and use of insecticides treated nets among the poorest populations in Kenya

    Directory of Open Access Journals (Sweden)

    Okungu Vincent

    2010-03-01

    Full Text Available Abstract Background Ensuring that the poor and vulnerable population benefit from malaria control interventions remains a challenge for malaria endemic countries. Until recently, ownership and use of insecticides treated nets (ITNs in most countries was low and inequitable, although coverage has increased in countries where free ITN distribution is integrated into mass vaccination campaigns. In Kenya, free ITNs were distributed to children aged below five years in 2006 through two mass campaigns. High and equitable coverage were reported after the campaigns in some districts, although national level coverage remained low, suggesting that understanding barriers to access remains important. This study was conducted to explore barriers to ownership and use of ITNs among the poorest populations before and after the mass campaigns, to identify strategies for improving coverage, and to make recommendations on how increased coverage levels can be sustained. Methods The study was conducted in the poorest areas of four malaria endemic districts in Kenya. Multiple data collection methods were applied including: cross-sectional surveys (n = 708 households, 24 focus group discussions and semi-structured interviews with 70 ITN suppliers. Results Affordability was reported as a major barrier to access but non-financial barriers were also shown to be important determinants. On the demand side key barriers to access included: mismatch between the types of ITNs supplied through interventions and community preferences; perceptions and beliefs on illness causes; physical location of suppliers and; distrust in free delivery and in the distribution agencies. Key barriers on the supply side included: distance from manufacturers; limited acceptability of ITNs provided through interventions; crowding out of the commercial sector and the price. Infrastructure, information and communication played a central role in promoting or hindering access. Conclusions Significant

  3. Utilization of insecticide-treated nets by under-five children in Nigeria: Assessing progress towards the Abuja targets

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    Sofola Olayemi T

    2008-07-01

    Full Text Available Abstract Background The Abuja target of increasing the proportion of people sleeping under insecticide-treated nets (ITNs to 60% by the year 2005, as one of the measures for malaria control in Africa, has generated an influx of resources for malaria control in several countries in the region. A national household survey conducted in 2005 by the Malaria Control Programme in Nigeria assessed the progress made with respect to ITN ownership and use among pregnant women and children under five years of age since 2000. The survey was the first nationally representative study of ITN use assessing progress towards the Abuja target amongst vulnerable groups. Population and Method A cross-sectional survey of a sample of 7,200 households, selected by a multistage stratified sampling technique from 12 randomly selected states from the six geopolitical zones of the country. Data collection was done during the malarious rainy season (October 2005 using a modified WHO Malaria Indicator Survey structured questionnaire about household ownership and utilization of mosquito nets (treated or untreated from household heads. Results Household ownership of any net was 23.9% (95% CI, 22.8%–25.1% and 10.1% for ITNs (95% CI, 9.2%–10.9%. Education, wealth index, presence of an under-five child in the household, family size, residence, and region by residence were predictive of ownership of any net. The presence of an under-five child in the household, family size, education, presence of health facility in the community, gender of household head, region by residence and wealth index by education predicted ITN ownership. Utilization of any net by children under-five was 11.5% (95% CI, 10.4%–12.6% and 1.7% (95% CI, 1.3%–2.2% for ITN. Predictors of use of any net among under-five children were fever in the previous two weeks, presence of health facility in the community, caregiver's education, residence, and wealth index by caregiver's education; while religion

  4. Reviewing the literature on access to prompt and effective malaria treatment in Kenya: implications for meeting the Abuja targets

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    Tetteh Gladys

    2009-10-01

    Full Text Available Abstract Background Effective case management is central to reducing malaria mortality and morbidity worldwide, but only a minority of those affected by malaria, have access to prompt effective treatment. In Kenya, the Division of Malaria Control is committed to ensuring that 80 percent of childhood fevers are treated with effective anti-malarial medicines within 24 hours of fever onset, but this target is largely unmet. This review aimed to document evidence on access to effective malaria treatment in Kenya, identify factors that influence access, and make recommendations on how to improve prompt access to effective malaria treatment. Since treatment-seeking patterns for malaria are similar in many settings in sub-Saharan Africa, the findings presented in this review have important lessons for other malaria endemic countries. Methods Internet searches were conducted in PUBMED (MEDLINE and HINARI databases using specific search terms and strategies. Grey literature was obtained by soliciting reports from individual researchers working in the treatment-seeking field, from websites of major organizations involved in malaria control and from international reports. Results The review indicated that malaria treatment-seeking occurs mostly in the informal sector; that most fevers are treated, but treatment is often ineffective. Irrational drug use was identified as a problem in most studies, but determinants of this behaviour were not documented. Availability of non-recommended medicines over-the-counter and the presence of substandard anti-malarials in the market are well documented. Demand side determinants of access include perception of illness causes, severity and timing of treatment, perceptions of treatment efficacy, simplicity of regimens and ability to pay. Supply side determinants include distance to health facilities, availability of medicines, prescribing and dispensing practices and quality of medicines. Policy level factors are around

  5. Application of Geospatial Information System to Assess the Effectiveness of the Mdg Target in Amac Metropolis-Abuja, Nigeria

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    T.T. Youngu

    2012-03-01

    Full Text Available This study aims to assess the effectiveness of the MDG target as it concerns, the state of education in Abuja Municipal Area Council (AMAC metropolis-Nigeria. One of the educational challenges faced in developing countries is how to know which school to send children to. This is mainly due to lack of detailed information as to, what school offers, what type of education, and at what fee. But with Geospatial Information Systems (GIS providing information about primary and secondary schools, Millennium Development Goals’ (MDGs’ vision of literacy for 15-24 year olds will be achieved. The development of geospatial database information system of primary and secondary schools for Garki and Wuse districts, Abuja, was embarked upon to provide timely, consistent and accurate information to decision makers for use in achieving this particular target of the Millennium Development Goals (MDGs. The delay in achievement of this target is not because governments in developing countries aren’t putting maximum effort, but because these governments are choosing to put the cart before the horse. Sustainable literacy level cannot be achieved without an effective framework charged with the collection, storage, query, analysis and management of the relevant data. What is needed is a system that provides government with data about all educational facilities within required proximity and determines availability of the required facilities. GIS is that decision making technology, which integrates ‘spatially referenced data’ with a problem solving environment. The method employed was to acquire both spatial and attribute data of every primary and secondary school in Garki and Wuse districts. The spatially referenced database was created using Microsoft Access 2007 and ArcGIS 9.2 software. The database was queried and the results of the queries analyzed. The results showed that 60% of the schools didn’t have access to Internet. Twenty-one (21 percent had no

  6. How to (or not to) … measure performance against the Abuja target for public health expenditure.

    Science.gov (United States)

    Witter, Sophie; Jones, Alex; Ensor, Tim

    2014-07-01

    In 2001, African heads of state committed 'to set a target of allocating at least 15% of our annual budget to the improvement of the health sector'. This target has since been used as a benchmark to hold governments accountable. However, it was never followed by a set of guidelines as to how it should be measured in practice. This article sets out some of the areas of ambiguity and argues for an interpretation which focuses on actual expenditure, rather than budgets (which are theoretical), and which captures areas of spending that are subject to government discretion. These are largely domestic sources, but include budget support, which is externally derived but subject to Ministry of Finance sectoral allocation. Theoretical and practical arguments in favour of this recommendation are recommended using a case study from Sierra Leone. It is recommended that all discretionary spending by government is included in the numerator and denominator when calculating performance against the target, including spending by all ministries on health, social health insurance payments, debt relief funds and budget support. Conversely, all forms of private payment and earmarked aid should be excluded. The authors argue that the target, while an important vehicle for tracking political commitment to the sector, should be assessed intelligently by governments, which have legitimate wider public finance objectives of maximizing overall social returns, and should be complemented by a wider range of indicators, to avoid distortions.

  7. Territory Abuja, Nigeria.

    African Journals Online (AJOL)

    country named Abuja 8,000 km2 in size greatly impacted on all spheres of lives of the ... E- mfilaba @yahoo.co.uk ... the urban poor - the job seekers in the ... gender roles as women started to become ... respect of peoples' views and culture and ... aged in-laws and children. The ..... Multi-national Corporations and individual.

  8. Assessment of Recreational Facilities in Federal Capital City, Abuja, Nigeria

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    Cyril Kanayo Ezeamaka

    2016-06-01

    Full Text Available Abuja Master Plan provided development of adequate Green Areas and other Recreational Facilities within the Federal Capital City (FCC, as part of its sustainability principles and provided for these recreational facilities within each neighborhood (FCDA, 1979. However, there have been several recent foul cries about the negative development of recreational facilities and the abuse of the Master Plan in the FCC.  The motivation for carrying out this study arose from the observation that recreational facilities in Phase 1 of the Federal Capital City Abuja are not clearly developed as intended by the policy makers and thus, the need to identify the recreational facilities in the Phase 1 of FCC and observe their level of development as well as usage. The field survey revealed that the Central Business District and Gazupe have higher numbers of recreational facilities with 45 and 56. While Wuse II (A08 and Wuse II (A07 Districts have lesser recreational facilities with 10 and 17. The field survey further revealed that all the districts in Phase 1 have over 35% cases of land use changes from recreational facilities to other use. The survey shows that over 65% of these recreational facilities are fully developed. The study also shows that just about 11% of the recreational sporting facilities were developed in line with the Abuja Master Plan in Phase 1. The study revealed that recreational facilities in Phase 1 of the FCC, Abuja has not being developed in compliance with the Abuja Master Plan.

  9. Tree Species Richness, Diversity, and Vegetation Index for Federal Capital Territory, Abuja, Nigeria

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    Aladesanmi D Agbelade

    2017-01-01

    Full Text Available This study was conducted to investigate the tree species richness and diversity of urban and periurban areas of the Federal Capital Territory (FCT, Abuja, Nigeria, and produce Normalized Difference Vegetation Index (NDVI for the territory. Data were collected from urban (Abuja city and periurban (Lugbe areas of the FCT using both semistructured questionnaire and inventory of tree species within green areas. In the study location, all trees with diameter at breast height (dbh ≥ 10 cm were identified; their dbh was measured and frequency was taken. The NDVI was calculated in ArcGIS 10.3 environment using standard formula. A cumulative total of twenty-nine (29 families were encountered within the FCT, with 27 occurring in Abuja city (urban centre and 12 in Lugbe (periurban centre of the FCT. The results of Shannon-Wiener diversity index (H′ for the two centres are 3.56 and 2.24 while Shannon’s maximum diversity index (Hmax is 6.54 (Abuja city and 5.36 (Lugbe for the urban (Abuja city and periurban (Lugbe areas of the Federal Capital Territory (FCT. The result of tree species evenness (Shannon’s equitability (EH index in urban and periurban centres was 0.54 and 0.42, respectively. The study provided baseline information on urban and periurban forests in the FCT of Nigeria, which can be used for the development of tree species database of the territory.

  10. High prevalence of HIV, chlamydia and gonorrhoea among men who have sex with men and transgender women attending trusted community centres in Abuja and Lagos, Nigeria

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    Babajide Keshinro

    2016-12-01

    Full Text Available Introduction: Sexually transmitted infection (STI and HIV prevalence have been reported to be higher amongst men who have sex with men (MSM in Nigeria than in the general population. The objective of this study was to characterize the prevalence of HIV, chlamydia and gonorrhoea in this population using laboratory-based universal testing. Methods: TRUST/RV368 represents a cohort of MSM and transgender women (TGW recruited at trusted community centres in Abuja and Lagos, Nigeria, using respondent-driven sampling (RDS. Participants undergo a structured comprehensive assessment of HIV-related risks and screening for anorectal and urogenital Chlamydia trachomatis and Neisseria gonorrhoeae, and HIV. Crude and RDS-weighted prevalence estimates with 95% confidence intervals (CIs were calculated. Log-binomial regression was used to explore factors associated with prevalent HIV infection and STIs. Results: From March 2013 to January 2016, 862 MSM and TGW (316 in Lagos and 546 in Abuja underwent screening for HIV, chlamydia and gonorrhoea at study enrolment. Participants’ median age was 24 years [interquartile range (IQR 21–27]. One-third (34.2% were identified as gay/homosexual and 65.2% as bisexual. The overall prevalence of HIV was 54.9%. After adjusting for the RDS recruitment method, HIV prevalence in Abuja was 43.5% (95% CI 37.3–49.6% and in Lagos was 65.6% (95% CI 54.7–76.5%. The RDS-weighted prevalence of chlamydia was 17.0% (95% CI 11.8–22.3% in Abuja and 18.3% (95% CI 11.1–25.4% in Lagos. Chlamydia infection was detected only at the anorectal site in 70.2% of cases. The RDS-weighted prevalence of gonorrhoea was 19.1% (95% CI 14.6–23.5% in Abuja and 25.8% (95% CI 17.1–34.6% in Lagos. Overall, 84.2% of gonorrhoea cases presented with anorectal infection only. Over 95% of STI cases were asymptomatic. In a multivariable model, increased risk for chlamydia/gonorrhoea was associated with younger age, gay/homosexual sexual orientation

  11. High prevalence of HIV, chlamydia and gonorrhoea among men who have sex with men and transgender women attending trusted community centres in Abuja and Lagos, Nigeria

    Science.gov (United States)

    Keshinro, Babajide; Crowell, Trevor A; Nowak, Rebecca G; Adebajo, Sylvia; Peel, Sheila; Gaydos, Charlotte A; Rodriguez-Hart, Cristina; Baral, Stefan D; Walsh, Melissa J; Njoku, Ogbonnaya S; Odeyemi, Sunday; Ngo-Ndomb, Teclaire; Blattner, William A; Robb, Merlin L; Charurat, Manhattan E; Ake, Julie

    2016-01-01

    Introduction Sexually transmitted infection (STI) and HIV prevalence have been reported to be higher amongst men who have sex with men (MSM) in Nigeria than in the general population. The objective of this study was to characterize the prevalence of HIV, chlamydia and gonorrhoea in this population using laboratory-based universal testing. Methods TRUST/RV368 represents a cohort of MSM and transgender women (TGW) recruited at trusted community centres in Abuja and Lagos, Nigeria, using respondent-driven sampling (RDS). Participants undergo a structured comprehensive assessment of HIV-related risks and screening for anorectal and urogenital Chlamydia trachomatis and Neisseria gonorrhoeae, and HIV. Crude and RDS-weighted prevalence estimates with 95% confidence intervals (CIs) were calculated. Log-binomial regression was used to explore factors associated with prevalent HIV infection and STIs. Results From March 2013 to January 2016, 862 MSM and TGW (316 in Lagos and 546 in Abuja) underwent screening for HIV, chlamydia and gonorrhoea at study enrolment. Participants’ median age was 24 years [interquartile range (IQR) 21–27]. One-third (34.2%) were identified as gay/homosexual and 65.2% as bisexual. The overall prevalence of HIV was 54.9%. After adjusting for the RDS recruitment method, HIV prevalence in Abuja was 43.5% (95% CI 37.3–49.6%) and in Lagos was 65.6% (95% CI 54.7–76.5%). The RDS-weighted prevalence of chlamydia was 17.0% (95% CI 11.8–22.3%) in Abuja and 18.3% (95% CI 11.1–25.4%) in Lagos. Chlamydia infection was detected only at the anorectal site in 70.2% of cases. The RDS-weighted prevalence of gonorrhoea was 19.1% (95% CI 14.6–23.5%) in Abuja and 25.8% (95% CI 17.1–34.6%) in Lagos. Overall, 84.2% of gonorrhoea cases presented with anorectal infection only. Over 95% of STI cases were asymptomatic. In a multivariable model, increased risk for chlamydia/gonorrhoea was associated with younger age, gay/homosexual sexual orientation and higher

  12. Adoption of Information and Communication Technologies (ICTs) by Agricultural Science and Extension Teachers in Abuja, Nigeria

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    Alabi, Olugbenga Omotayo

    2016-01-01

    This study examined adoption of Information and Communication Technologies (ICTs) by agricultural science and extension teachers in Abuja, Nigeria. Specifically, the objectives are to: identify the background and demographic characteristics of agricultural science and extension teachers in the study area; examine the factors influencing adoption…

  13. Study Identifies New Lymphoma Treatment Target

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    NCI researchers have identified new therapeutic targets for diffuse large B-cell lymphoma. Drugs that hit these targets are under clinical development and the researchers hope to begin testing them in clinical trials of patients with DLBCL.

  14. Clinical and demographic characteristics of women with intrauterine adhesion in abuja, Nigeria.

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    Efetie, Efena R; Umezulike, Augustine C; Okafor, Ugochukwu V

    2012-01-01

    Objective. Infertility menstrual abnormalities continue to constitute a significant bulk of gynecological consultation in Africa. Both of these problems are sometimes traced to intrauterine adhesions which are preventable in the majority of cases. Study Design. A retrospective analysis of intrauterine adhesions at the National Hospital Abuja, Nigeria, was carried out, covering the period from 1st September 1999 to 1st September 2004. A total of 72 cases were analyzed. Statical analysis was done using X(2). Results. The incidence of intrauterine adhesions was 1.73% of new patients. Mean age ± SD was 29.97 ± 4.82 years. Patients who were Para 0 to 1 constituted 81.9% of the total. Intrauterine adhesions significantly (P < 0.02) occurred in nulliparae. The majority (68%) were educated only up to secondary level which was significant (P < 0.05). Menstrual abnormalities were present in 90.3%. The commonest predisposing factor identified was a history of dilatation and curettage or uterine evacuation. Conclusion. Intrauterine adhesions are associated with lower educational status and low parity. Increasing educational targets nationally, poverty alleviation, nationwide retraining in manual vacuum aspiration, and wider application of this technique are recommended.

  15. Clinical and Demographic Characteristics of Women with Intrauterine Adhesion in Abuja, Nigeria

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    Efena R. Efetie

    2012-01-01

    Full Text Available Objective. Infertility menstrual abnormalities continue to constitute a significant bulk of gynecological consultation in Africa. Both of these problems are sometimes traced to intrauterine adhesions which are preventable in the majority of cases. Study Design. A retrospective analysis of intrauterine adhesions at the National Hospital Abuja, Nigeria, was carried out, covering the period from 1st September 1999 to 1st September 2004. A total of 72 cases were analyzed. Statical analysis was done using 2. Results. The incidence of intrauterine adhesions was 1.73% of new patients. Mean age ± SD was 29.97±4.82 years. Patients who were Para 0 to 1 constituted 81.9% of the total. Intrauterine adhesions significantly (<0.02 occurred in nulliparae. The majority (68% were educated only up to secondary level which was significant (<0.05. Menstrual abnormalities were present in 90.3%. The commonest predisposing factor identified was a history of dilatation and curettage or uterine evacuation. Conclusion. Intrauterine adhesions are associated with lower educational status and low parity. Increasing educational targets nationally, poverty alleviation, nationwide retraining in manual vacuum aspiration, and wider application of this technique are recommended.

  16. evaluation of the environmental noise levels in abuja municipality ...

    African Journals Online (AJOL)

    10-14. Noise pollution has been proven to aggravate chronic illnesses like hypertension and other cardiopulmonary diseases.8, 9 .... health hazard of different classes and magnitude ... sound levels in Abuja are fairly good in contrast to the .... Noise and Health 2012; 14(59): ... Information Technologies in Environmental.

  17. Notes on the History of Abuja, Central Nigeria

    OpenAIRE

    THOMAS-EMEAGWALI, Gloria

    1989-01-01

    This paper is concerned with aspects of the history of Abuja in the Central Nigerian region -- an area designated as the new Federal capital of Nigeria. We examine some aspects of inter-group relations and then reflect on technological and economic development in the area. In the course of discussion, we reflect on aspects of iron technology, textile, leather processing and pottery and comment on issues related to trade in the region.

  18. Isolation and identification of fungi associated with spoilt fruits vended in Gwagwalada market, Abuja, Nigeria.

    Science.gov (United States)

    Mailafia, Samuel; Okoh, God'spower Richard; Olabode, Hamza Olatunde K; Osanupin, Ramatu

    2017-04-01

    Annual reports have shown that 20% of fruits and vegetables produced are lost to spoilage. This study was undertaken to isolate and identify fungi that are associated with spoilt fruits commonly sold in Gwagwalada market, Abuja, and recommend appropriate control measure. The study was conducted in Gwagwalada metropolis, Gwagwalada Area Council of the Federal Capital Territory, Abuja, Nigeria. A total of 100 spoilt fruits which include pawpaw (Carica papaya), orange (Citrus sinensis), tomato (Lycopersicon esculentum), pineapple (Ananas comosus), and watermelon (Citrullus vulgaris) were purchased and examined for the presence of fungal organisms using standard methods. The data collected were analyzed using simple descriptive statistics (frequency and mean) and analysis of variance (pfrequency of 38%. Fusarium avenaceum followed with the frequency of occurrence of 31% in fruits such as pineapple, watermelon, oranges, pawpaw, and tomatoes while Penicillium digitatum and Rhizopus stolonifer had the least frequency of 4% each in tomato; and orange and tomato, respectively. Other fungal species were identified as yeast (Saccharomyces species) (10%), Fusarium solani (8%), and Aspergillus flavus (5%). The highest prevalence rate was 70% of A. niger from orange followed by F. avenaceum of which 65% isolates were recovered from pawpaw. Other fungal organisms such as yeast (Saccharomyces species), P. digitatum and R. stolonifer were isolated with varying prevalence (40%, 20%, and 5%) from watermelon, tomato, and orange, respectively. However, there was no significant difference in the fungal load of the various fruits studied (analysis of variance=478.2857, p<0.05, F=4.680067 and df=34). The pathogenic fungi species associated with fruits spoilage in this study are of economical and public health significance. A. niger causes black mold in certain fruits and vegetables. Some strains of A. niger have been reported to produce potent mycotoxins called ochratoxins that can be

  19. Using Click Chemistry to Identify Potential Drug Targets in Plasmodium

    Science.gov (United States)

    2015-04-01

    AWARD NUMBER: W81XWH-13-1-0429 TITLE: Using "Click Chemistry" to Identify Potential Drug Targets in Plasmodium PRINCIPAL INVESTIGATOR: Dr. Purnima...SUBTITLE Sa. CONTRACT NUMBER W81XWH-1 3-1-0429 Using "Click Chemistry" to Identify Potential Drug Targets in Plasmodium 5b. GRANT NUMBER 5c. PROGRAM...Release; Distribution Unlimited 13. SUPPLEMENTARY NOTES 14. ABSTRACT Sporozo ite infection of the liver is the first obl igate step of the Plasmodium

  20. Identifying unexpected therapeutic targets via chemical-protein interactome.

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    Lun Yang

    Full Text Available Drug medications inevitably affect not only their intended protein targets but also other proteins as well. In this study we examined the hypothesis that drugs that share the same therapeutic effect also share a common therapeutic mechanism by targeting not only known drug targets, but also by interacting unexpectedly on the same cryptic targets. By constructing and mining an Alzheimer's disease (AD drug-oriented chemical-protein interactome (CPI using a matrix of 10 drug molecules known to treat AD towards 401 human protein pockets, we found that such cryptic targets exist. We recovered from CPI the only validated therapeutic target of AD, acetylcholinesterase (ACHE, and highlighted several other putative targets. For example, we discovered that estrogen receptor (ER and histone deacetylase (HDAC, which have recently been identified as two new therapeutic targets of AD, might already have been targeted by the marketed AD drugs. We further established that the CPI profile of a drug can reflect its interacting character towards multi-protein sets, and that drugs with the same therapeutic attribute will share a similar interacting profile. These findings indicate that the CPI could represent the landscape of chemical-protein interactions and uncover "behind-the-scenes" aspects of the therapeutic mechanisms of existing drugs, providing testable hypotheses of the key nodes for network pharmacology or brand new drug targets for one-target pharmacology paradigm.

  1. Stroke risk factors, subtypes, and 30‑day case fatality in Abuja, Nigeria

    African Journals Online (AJOL)

    Stroke risk factors, subtypes, and 30‑day case fatality in Abuja, Nigeria. ... as well as brain computerized tomography, magnetic resonance imaging, and carotid ... We also assessed case fatality and functional outcome at 30 days after stroke.

  2. review article urinary tract infections in a tertiary hospital in abuja ...

    African Journals Online (AJOL)

    boaz

    AFRICAN JOURNAL OF CLINICAL AND EXPERIMENTAL MICROBIOLOGY SEPTEMBER ... Department of Medical Microbiology and Parasitology, National Hospital Abuja, P.M.B 425, Nigeria. ..... reproductive biology2011;156(2):131–6. 34.

  3. Identifying targets for quality improvement in hospital antibiotic prescribing

    NARCIS (Netherlands)

    Spreuwel, P.C. van; Blok, H.; Langelaar, M.F.; Kullberg, B.J.; Mouton, J.W.; Natsch, S.S.

    2015-01-01

    OBJECTIVES: To audit antibiotic use in a university hospital and to identify targets for quality improvement in a setting with low antibiotic use and resistance rates. METHODOLOGY: A point-prevalence survey (PPS), using a patient-based audit tool for antibiotic use, was executed in the Radboud Unive

  4. Identifying problematic drugs based on the characteristics of their targets

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    Tiago Jose eDa Silva Lopes

    2015-09-01

    Full Text Available Identifying promising compounds during the early stages of drug development is a major challenge for both academia and the pharmaceutical industry. The difficulties are even more pronounced when we consider multi-target pharmacology, where the compounds often target more than one protein, or multiple compounds are used together. Here, we address this problem by using machine learning and network analysis to process sequence and interaction data from human proteins to identify promising compounds. We used this strategy to identify properties that make certain proteins more likely to cause harmful effects when targeted; such proteins usually have domains commonly found throughout the human proteome. Additionally, since currently marketed drugs hit multiple targets simultaneously, we combined the information from individual proteins to devise a score that quantifies the likelihood of a compound being harmful to humans. This approach enabled us to distinguish between approved and problematic drugs with an accuracy of 60%¬–70%. Moreover, our approach can be applied as soon as candidate drugs are available, as demonstrated with predictions for more than 5000 experimental drugs. These resources are available at http://sourceforge.net/projects/psin/.

  5. Interaction profiling identifies the human nuclear exosome targeting complex

    DEFF Research Database (Denmark)

    Lubas, Michal Szymon; Christensen, Marianne Spangsberg; Kristiansen, Maiken Søndergaard

    2011-01-01

    of a similar activator(s) in humans remains elusive. By establishing an interaction network of the human nuclear exosome, we identify the trimeric Nuclear Exosome Targeting (NEXT) complex, containing hMTR4, the Zn-knuckle protein ZCCHC8, and the putative RNA binding protein RBM7. ZCCHC8 and RBM7 are excluded...... to nucleoli. Our results suggest that human nuclear exosome degradation pathways comprise modules of spatially organized cofactors that diverge from the yeast model....

  6. Using Click Chemistry to Identify Potential Drug Targets in Plasmodium

    Science.gov (United States)

    2016-06-01

    both enzymes are expressed cytoplasmically in sporozoites and liver stages. Using a specific and potent inhibitor of Plasmodium PKG and inhibitor... originally planned. Therefore, we modified our approach so that we could still fulfill the objective of identifying Tsp’s target in sporozoites. To fulfill...essential Ca(2)(+) signals at key decision points in the life cycle of malaria parasites. PLoS Biol 12: e1001806. 2. Falae A, Combe A, Amaladoss A

  7. Identifying molecular targets of lifestyle modifications in colon cancer prevention

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    Molly Marie Derry

    2013-05-01

    Full Text Available One in four deaths in the United States is cancer-related, and colorectal cancer (CRC is the second leading cause of cancer-associated deaths. Screening strategies are utilized but have not reduced disease incidence or mortality. In this regard, there is an interest in cancer preventive strategies focusing on lifestyle intervention, where specific etiologic factors involved in cancer initiation, promotion, and progression could be targeted. For example, exposure to dietary carcinogens, such as nitrosamines and polycyclic aromatic hydrocarbons influences colon carcinogenesis. Furthermore, dietary deficiencies could alter sensitivity to genetic damage and influence carcinogen metabolism contributing to CRC. High alcohol consumption increases the risk of mutations including the fact that acetaldehyde, an ethanol metabolite, is classified as a group 1 carcinogen. Tobacco smoke exposure is also a risk factor for cancer development; ~20% of CRCs are associated with smoking. Additionally, obese patients have a higher risk of cancer development, which is further supported by the fact that physical activity decreases CRC risk by 55%. Similarly, chronic inflammatory conditions also increase the risk of CRC development. Moreover, the circadian clock alters digestion and regulates other biochemical, physiological and behavioral processes that could positively influence CRC. Taken together, colon carcinogenesis involves a number of etiological factors, and therefore, to create effective preventive strategies, molecular targets need to be identified and beleaguered prior to disease progression. With this in mind, the following is a comprehensive review identifying downstream target proteins of the above lifestyle risk factors, which are modulated during colon carcinogenesis and could be targeted for CRC prevention by novel agents including phytochemicals.

  8. The pattern of paediatric HIV/AIDS as seen at the National Hospital Abuja Nigeria.

    Science.gov (United States)

    Oniyangi, O; Awani, B; Iregbu, K C

    2006-12-01

    Paediatric HIV/AIDS has become a significant cause of mortality and morbidity in our environment. The objective of this paper is to determine the mode of transmission, clinical presentations and outcome of hospital admissions in children with Paediatric HIV/AIDS at the National Hospital Abuja Nigeria. A retrospective study of children with Paediatric HIV/AIDS admitted into the hospital from January December 2000 was done. Screening for HIV infection was based on clinical criteria as recommended by WHO except in 3 children with previously diagnosed HIV seropositivity. One positive ELISA and one positive Western Blot assay diagnosed HIV seropositivity. Forty-three HIV positive children aged six weeks to nine years (mean 16.5 months, SD 26.32) were admitted into the Paediatric unit (exclusive of the newborn unit) of the hospital, accounting for 5.7% of all admissions into the unit. There were 35 infants (81.4%). There were 18 males and 25 females (male: female ratio 1:0.72). The presumed modes of transmission were mother to child transmission 40(93.02%), blood transfusion 2 (4.6%) and an unidentified route 1 (2.3%). All parents were in the reproductive age group and there were 6 discordant couples identified (mother HIV positive, father HIV negative). Common presenting symptoms were fever 16 (37.2.8%), diarrhoea 13 (30.2%), difficult/fast breathing 12 (27.9%) and vomiting 8 (18.6%), while clinical signs were crepitations in the lungs 27 (62.7%), pallor 22 (51.2%), oral thrush 20 (46.5%), hepatomegaly 18 (41.9%), and dehydration 16 (37.2%). Admitting diagnoses were pneumonia 26 (60.5%), septicaemia 4 (9.3%), diarrhoea with dehydration, intestinal obstruction and malnutrition 2 (4.7%) each. There were 14 deaths (mortality rate 32.6%); accounting for 28.57% of total deaths in the paediatric unit during the period. Thirteen (13) (92.8%) deaths occurred in children aged 2 years old and below. The greatest contributors to mortality were pneumonia 10 (71.4%) and septicaemia

  9. Identifying microRNAs and transcript targets in Jatropha seeds.

    Directory of Open Access Journals (Sweden)

    Vanessa Galli

    Full Text Available MicroRNAs, or miRNAs, are endogenously encoded small RNAs that play a key role in diverse plant biological processes. Jatropha curcas L. has received significant attention as a potential oilseed crop for the production of renewable oil. Here, a sRNA library of mature seeds and three mRNA libraries from three different seed development stages were generated by deep sequencing to identify and characterize the miRNAs and pre-miRNAs of J. curcas. Computational analysis was used for the identification of 180 conserved miRNAs and 41 precursors (pre-miRNAs as well as 16 novel pre-miRNAs. The predicted miRNA target genes are involved in a broad range of physiological functions, including cellular structure, nuclear function, translation, transport, hormone synthesis, defense, and lipid metabolism. Some pre-miRNA and miRNA targets vary in abundance between the three stages of seed development. A search for sequences that produce siRNA was performed, and the results indicated that J. curcas siRNAs play a role in nuclear functions, transport, catalytic processes and disease resistance. This study presents the first large scale identification of J. curcas miRNAs and their targets in mature seeds based on deep sequencing, and it contributes to a functional understanding of these miRNAs.

  10. Anti-schistosomal Intervention Targets Identified by Lifecycle Transcriptomic Analyses

    Science.gov (United States)

    Fitzpatrick, Jennifer M.; Peak, Emily; Perally, Samirah; Chalmers, Iain W.; Barrett, John; Yoshino, Timothy P.; Ivens, Alasdair C.; Hoffmann, Karl F.

    2009-01-01

    Background Novel methods to identify anthelmintic drug and vaccine targets are urgently needed, especially for those parasite species currently being controlled by singular, often limited strategies. A clearer understanding of the transcriptional components underpinning helminth development will enable identification of exploitable molecules essential for successful parasite/host interactions. Towards this end, we present a combinatorial, bioinformatics-led approach, employing both statistical and network analyses of transcriptomic data, for identifying new immunoprophylactic and therapeutic lead targets to combat schistosomiasis. Methodology/Principal Findings Utilisation of a Schistosoma mansoni oligonucleotide DNA microarray consisting of 37,632 elements enabled gene expression profiling from 15 distinct parasite lifecycle stages, spanning three unique ecological niches. Statistical approaches of data analysis revealed differential expression of 973 gene products that minimally describe the three major characteristics of schistosome development: asexual processes within intermediate snail hosts, sexual maturation within definitive vertebrate hosts and sexual dimorphism amongst adult male and female worms. Furthermore, we identified a group of 338 constitutively expressed schistosome gene products (including 41 transcripts sharing no sequence similarity outside the Platyhelminthes), which are likely to be essential for schistosome lifecycle progression. While highly informative, statistics-led bioinformatics mining of the transcriptional dataset has limitations, including the inability to identify higher order relationships between differentially expressed transcripts and lifecycle stages. Network analysis, coupled to Gene Ontology enrichment investigations, facilitated a re-examination of the dataset and identified 387 clusters (containing 12,132 gene products) displaying novel examples of developmentally regulated classes (including 294 schistosomula and

  11. Managing Pre-Construction and Construction Risks on Project Sites in Abuja-Nigeria

    Directory of Open Access Journals (Sweden)

    Jimoh R.A.

    2016-03-01

    Full Text Available Managing risks in construction projects has been acknowledged as a key direction process for the purposes of attaining the project goal in terms of time, cost, quality, safety, and environmental sustainability. Hence, the paper evaluated pre-construction and construction risks on active project sites in Abuja-Nigeria. This was achieved using survey method through the self-administration of 35 questionnaires to the professionals handling the 35 identified projects being undertaking at that time. Results showed that errors and omissions in design and improperly defined project scope had mean score values of 3.03 and 2.54, respectively, were the construction risks most experienced by the professionals during pre-construction. On the other hand, fluctuation in market prices and delays with mean score values of 3.14 and 2.74, respectively, were the construction risks experienced by the professionals during the construction phase of the projects. It is recommended that procurement methods such as construction management should be adopted and stakeholders should keep to their own side of the bargain to avoid unnecessary delays.

  12. The Impact of Home Environment Factors on Academic Performance of Senior Secondary School Students in Garki Area District, Abuja - Nigeria

    Directory of Open Access Journals (Sweden)

    L. T. Dzever

    2015-12-01

    Full Text Available The study examined the impact of home environment factors on the academic performance of public secondary school students in Garki Area District, Abuja, Nigeria. The stratified sampling technique was used to select 300 students from six public schools, while the simple random sampling technique was used to administer the questionnaire. The study utilized a descriptive survey research design for the study. Also, data on student’s academic performance was obtained from student’s scores in four selected school subjects. Data obtained was analyzed using descriptive and inferential statistical techniques; Pearson Product Moment Correlation and Multiple regression analysis (ANOVA. The results result revealed a positive and significant relationship between permissive patenting style with academic performance (p0.05. Also, the result from the study identified income, educational background and occupational level as well as permissive parenting style as the main predictive variables influencing students’ academic performance.

  13. Gene expression profiling: can we identify the right target genes?

    Directory of Open Access Journals (Sweden)

    J. E. Loyd

    2008-12-01

    Full Text Available Gene expression profiling allows the simultaneous monitoring of the transcriptional behaviour of thousands of genes, which may potentially be involved in disease development. Several studies have been performed in idiopathic pulmonary fibrosis (IPF, which aim to define genetic links to the disease in an attempt to improve the current understanding of the underlying pathogenesis of the disease and target pathways for intervention. Expression profiling has shown a clear difference in gene expression between IPF and normal lung tissue, and has identified a wide range of candidate genes, including those known to encode for proteins involved in extracellular matrix formation and degradation, growth factors and chemokines. Recently, familial pulmonary fibrosis cohorts have been examined in an attempt to detect specific genetic mutations associated with IPF. To date, these studies have identified families in which IPF is associated with mutations in the gene encoding surfactant protein C, or with mutations in genes encoding components of telomerase. Although rare and clearly not responsible for the disease in all individuals, the nature of these mutations highlight the importance of the alveolar epithelium in disease pathogenesis and demonstrate the potential for gene expression profiling in helping to advance the current understanding of idiopathic pulmonary fibrosis.

  14. Virtual target screening to rapidly identify potential protein targets of natural products in drug discovery

    Directory of Open Access Journals (Sweden)

    Yuri Pevzner

    2014-05-01

    Full Text Available Inherent biological viability and diversity of natural products make them a potentially rich source for new therapeutics. However, identification of bioactive compounds with desired therapeutic effects and identification of their protein targets is a laborious, expensive process. Extracts from organism samples may show desired activity in phenotypic assays but specific bioactive compounds must be isolated through further separation methods and protein targets must be identified by more specific phenotypic and in vitro experimental assays. Still, questions remain as to whether all relevant protein targets for a compound have been identified. The desire is to understand breadth of purposing for the compound to maximize its use and intellectual property, and to avoid further development of compounds with insurmountable adverse effects. Previously we developed a Virtual Target Screening system that computationally screens one or more compounds against a collection of virtual protein structures. By scoring each compound-protein interaction, we can compare against averaged scores of synthetic drug-like compounds to determine if a particular protein would be a potential target of a compound of interest. Here we provide examples of natural products screened through our system as we assess advantages and shortcomings of our current system in regards to natural product drug discovery.

  15. Cancer therapeutic target genes identified on chromosome 20q

    Directory of Open Access Journals (Sweden)

    Editorial Office

    2016-08-01

    Full Text Available An integrated quantitative genome data analysis was recently able to pinpoint 18 genes on human chromosome 20q that could potentially serve as novel molecular targets for cancer therapy. Researchers Antoine M Snijders and Jian-Hua Mao from Lawrence Berkeley National Laboratory’s Biological Systems and Engineering Division in Berkeley, California, United States, in their study published by the journal Advances in Modern Oncology Research (AMOR sought to compare the amounts of individual mRNAs – messenger RNAs that specify the amino acid sequence of the protein products of gene expression – in cancerous human tissues with corresponding normal tissues. The duo conducted a meta-analysis of genes on chromosome 20q that are found to be consistently upregulated across different human tumor types, while collecting gene transcript data of normal and tumor tissues across 11 different tumor types including brain, breast, colon, gastric, head and neck, liver, lung, ovarian, cervix, pancreas, and prostate cancers. “We calculated the differential expression of all 301 genes present on chromosome 20q for which gene transcript data was available. We then filtered for genes that were upregulated in tumors by at least 1.5 fold (p < 0.05 in seven or more tumor types,” they said. The resulting analysis identified 18 genes – some such as AURKA, UBE2C, TPX2, FAM83D, ZNF217, SALL4 and MMP9 have been previously known to potentially cause cancer. The 18-gene signature is revealed by the study to have robustly elevated levels across human cancers. “We observed significant association of our signature with disease-free survival in all 18 independent data… These data indicated that our signature is broadly predictive for disease-free survival, independent of tumor type,” the researchers said. In certain cases, Snijders and Mao found that increased gene expression was associated with better prognosis. “For example, the increased expressions of MMP9 and

  16. Macrosomic births in abuja: A case–control study of predisposing ...

    African Journals Online (AJOL)

    Macrosomic births in abuja: A case–control study of predisposing factors and early ... Independent predictors of macrosomia were parental high social class (P ... between the macrosomic group (2.5%) and the control group (0.8%) was not ...

  17. Identifying drug-target proteins based on network features

    Institute of Scientific and Technical Information of China (English)

    ZHU MingZhu; GAO Lei; LI Xia; LIU ZhiCheng

    2009-01-01

    Proteins rarely function in isolation Inside and outside cells, but operate as part of a highly Intercon-nected cellular network called the interaction network. Therefore, the analysis of the properties of drug-target proteins in the biological network is especially helpful for understanding the mechanism of drug action In terms of informatice. At present, no detailed characterization and description of the topological features of drug-target proteins have been available in the human protein-protein interac-tion network. In this work, by mapping the drug-targets in DrugBank onto the interaction network of human proteins, five topological indices of drug-targets were analyzed and compared with those of the whole protein interactome set and the non-drug-target set. The experimental results showed that drug-target proteins have higher connectivity and quicker communication with each other in the PPI network. Based on these features, all proteins In the interaction network were ranked. The results showed that, of the top 100 proteins, 48 are covered by DrugBank; of the remaining 52 proteins, 9 are drug-target proteins covered by the TTD, Matador and other databases, while others have been dem-onstrated to be drug-target proteins in the literature.

  18. Identifying drug-target proteins based on network features

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Proteins rarely function in isolation inside and outside cells, but operate as part of a highly intercon- nected cellular network called the interaction network. Therefore, the analysis of the properties of drug-target proteins in the biological network is especially helpful for understanding the mechanism of drug action in terms of informatics. At present, no detailed characterization and description of the topological features of drug-target proteins have been available in the human protein-protein interac- tion network. In this work, by mapping the drug-targets in DrugBank onto the interaction network of human proteins, five topological indices of drug-targets were analyzed and compared with those of the whole protein interactome set and the non-drug-target set. The experimental results showed that drug-target proteins have higher connectivity and quicker communication with each other in the PPI network. Based on these features, all proteins in the interaction network were ranked. The results showed that, of the top 100 proteins, 48 are covered by DrugBank; of the remaining 52 proteins, 9 are drug-target proteins covered by the TTD, Matador and other databases, while others have been dem- onstrated to be drug-target proteins in the literature.

  19. Prevalence and correlates of violence against female sex workers in Abuja, Nigeria.

    Science.gov (United States)

    Fawole, Olufunmilayo I; Dagunduro, Abosede T

    2014-06-01

    Few studies in Africa provide detailed descriptions of the vulnerabilities of female sex workers (FSWs) to violence. To document the prevalence and types of violence experienced by FSWs, identify the risk factors of experiencing violence to women (VAW) and the perpetrators of these acts. An analytical cross sectional survey of 305 brothel-based FSWs and in-depth interview of 20 chairpersons residing in brothels in Abuja, Nigeria was done. The prevalence of VAW six months preceding the survey was 52.5%. Sexual violence was the commonest type (41.9%) of violence experienced, followed by economic (37.7%), physical violence (35.7%) and psychological (31.9%). The main perpetrators of sexual violence were clients (63.8%) and brothel management (18.7%). Sexual violence was significantly more experienced (aOR 2.23; 95%CI 1.15-4.36) by older FSWs than their younger counterparts, by permanent brothel residents (aOR 2.08; 95%CI 1.22-3.55) and among those who had been in the sex industry for more than five years (aOR 2.01; 95%CI 0.98-4.10). Respondents with good knowledge levels of types of violence were less vulnerable to physical violence (aOR 0.45; 95%CI 0.26-0.77). Psychological violence was more likely among FSWs who smoked (aOR 2.16; 95%CI 1.26-3.81). Risk of economic violence decreased with educational levels (aOR 0.54; 95%CI 0.30-0.99 and aOR 0.42; 95%CI 0.22-0.83 for secondary and post secondary respectively). Consequences of the violence included sexually transmitted infections (20%) and HIV (8.0%). Interventions that educate FSWs on their rights and enable them avoid violence are urgently required. Young women need economic and educational empowerments to enable them avoid sex work.

  20. Abuja declaration calls for action against hazardous traditional practices.

    Science.gov (United States)

    1990-05-01

    The UN Economic Commission for Africa organized a conference in Abuja, Nigeria, last November to review the "Role of Women in Africa in the 1990s" as a follow-up of the "Arusha Strategies" of 1984. Among topics examined were harmful traditional practices, such as early marriage and pregnancy, female circumcision, nutritional taboos, inadequate child spacing and unprotected delivery, which are still found to be current realities in many African countries. These practices often inflict permanent physical, psychological, and emotional damage, even death, and little progress has been achieved in the abolition, the Declaration states. The lives of women in Africa are dominated by traditions. Certain attitudes, structures, and traditional practices, such as female circumcision and nutritional taboos that have harmful effects on the health of women and children, have rarely been officially surveyed. They have not been fully acknowledged by policy makers and opinion leaders, nor have effective steps to stop them been given precedence in health development planning. There is need for action at national as well as subregional and regional levels. Action at the national levels means that: national research institutes should undertake in-depth research on various traditional practices and their effects on women; functional literacy campaigns should sensitize parents and disseminate information on the harmful effects of circumcision, childhood marriage and early pregnancy; guidance and counseling should be provided to adolescent girls as well as to parents to make them understand the harmful physical, social, and mental effects of some traditional practices; religious leaders, traditional rulers, women's organizational, professional bodies and others should act as pressure groups in promoting efforts against harmful practices through traditional and modern means of communication, dissemination of information, and other appropriate ways of communication; and legislative and

  1. 10 years of Vertigo Clinic at National Hospital Abuja, Nigeria: what have we learned?

    Science.gov (United States)

    Olusesi, Abiodun D; Abubakar, J

    2016-11-01

    The clinician's major role in management of the dizzy patient involves determining what dizziness is vertigo, and what vertigo is of central or peripheral origin. These demand attention to details of history, otolaryngological workup including vestibular assessment, and often use of diagnostic and management algorithms. There is paucity of published reports of the management outcomes of peripheral vestibular diseases from Africa. Two tertiary care otologist-led dedicated vertigo clinics are located in Abuja, Nigeria. A prospective, non-randomized study of patients presenting with features of peripheral vestibular diseases attending the National Hospital Abuja Nigeria (between May 2005 and April 2014) and CSR Otologics Specialist Clinics (May 2010 to April 2014) was carried out. Both institutions adopted the same diagnostic and management protocols. Data extracted from anonymized databases created for this study include age, sex, vertigo duration (acute 12 weeks), dizziness handicap inventory score at presentation and at subsequent visits, otological and vestibular findings, ice-water caloric testing results, other investigation outcomes, treatments offered and outcomes. 561/575 (97.5 %) of the cases recorded had peripheral vestibular disease. The male-to-female ratio was 290:271. The mean age of the subjects was 44.7 years. Duration of vertigo at presentation was acute in 278 subjects and chronic in 283 subjects. Identifiable clinical diagnostic groups include BPPV (n = 200), Meniere's disease (n = 189), cervicogenic vertigo (n = 35), labyrinthitis (n = 32), Migraine-associated vertigo (MAV) (n = 32), cholesteatoma/perilymph Fistula (n = 10), climacteric vertigo (n = 8) and unclassified vertigo (n = 55). Migraine-associated vertigo recorded the highest DHI score (95 % CI 75 ± 4.3), followed by cholesteatoma/perilymph fistula (95 % CI 72 ± 6.1) and labyrinthitis (95 % CI 62 ± 1.9). Pure tone audiometry (95 % CI 67.3 ± 3

  2. Bacterial isolates associated with pelvic inflammatory disease among female patients attending some hospitals in abuja, Nigeria.

    Science.gov (United States)

    Spencer, T H I; Umeh, P O; Irokanulo, E; Baba, M M; Spencer, B B; Umar, A I; Ardzard, S A; Oderinde, S; Onoja, O

    2014-01-01

    Pelvic inflammatory disease refers to any infection in the female lower reproductive tract that spreads to the upper reproductive tract. The disease comprises a spectrum of inflammatory disorders of the upper female genital tract, including any combination of endometritis, salpingitis, tubo-ovarian abscess and pelvic peritonitis. PID is not a notifiable disease in most countries, so accurate statistics are not available. This situation is not in any way different here in Nigeria and more so in the Federal Capital Territory, Abuja where this research was conducted, there had never been any published report so far on PID. It therefore became pertinent that such studies be carried out to evaluate the bacterial organisms which may be associated with the disease in this part of Nigeria so that health care providers could take a better look at this affliction in women. Endocervical swabs totalling 100 were aseptically collected from patients with confirmed Pelvic Inflammatory Disease (PID), attending some hospitals in Abuja, Nigeria for detection of bacterial pathogens based on cultural and biochemical characterisation tests. Antibiogram was also conducted on the identified bacterial isolates. Out of the 100 samples analysed, 43% yielded pure cultures of bacterial isolates, 2% yielded mixed cultures while no bacterial growths were recorded from the remaining 55% samples. Organisms encountered were Staphylococcus aureus (16%), Escherichia coli (10%), Streptococcus faecalis (8%), Pseudomonas aeruginosa (4%), Streptococcus pyogenes (3%), Klebsiella pneumoniae (3%), Proteus rettgeri (2%) and Proteus mirabilis (1%). The highest percentage occurrence of pathogenic isolates was observed in polygamous married patients (90%). The age group most affected falls within the mean age 30.5 years (68%) while the least affected group falls within the mean age 40.5 years (5%). There was a significant difference in the acquisition of PID in relation to marital status (P disease with

  3. Identifying exposure targets for treatment of staphylococcal pneumonia with ceftobiprole.

    Science.gov (United States)

    Rodvold, Keith A; Nicolau, David P; Lodise, Thomas P; Khashab, Mohammed; Noel, Gary J; Kahn, James B; Gotfried, Mark; Murray, Sara A; Nicholson, Susan; Laohavaleeson, Somvadee; Tessier, Pamela R; Drusano, G L

    2009-08-01

    Ceftobiprole is a cephalosporin with potent activity against methicillin (meticillin)-resistant Staphylococcus aureus (MRSA). In order to treat patients with severe staphylococcal pneumonia, it is important to understand the drug exposure required to mediate the killing of multiple log(10) cells in a preclinical-infection model. We measured drug exposure in terms of the percentage of penetration of the drug into epithelial lining fluid (ELF) and in terms of the time for which the drug concentration was above the MIC (time>MIC) in plasma and ELF. In a murine model of staphylococcal pneumonia, we demonstrated that ceftobiprole penetrated into ELF from the plasma at a median level of nearly 69% (25th to 75th percentile range, 25 to 187%), as indexed to the ratio of values for the area under the concentration-time curve in ELF and plasma. The total-drug times>MIC in ELF that were required to kill 1 log(10) and 2 log(10) CFU/g of lung tissue were 15% and 25% of the dosing interval. We also examined the penetration of ELF by ceftobiprole in volunteers, demonstrating mean and median penetration percentages of 25.5% and 15.3%, respectively (25th to 75th percentile range, 8 to 30%). Attainment rates were calculated for kill targets of 1 log(10) and 2 log(10) CFU/g, taken from the murine model, but using the volunteer ceftobiprole ELF penetration data. The standard dose for ceftobiprole is 0.5 g every 8 h as a 2-h infusion. The attainment rates remained above 90% for 1-log(10) and 2-log(10) CFU/g kill targets at MICs of 1 and 0.5 mg/liter, respectively. Taking the expectation over the distribution of ceftobiprole MICs for 4,958 MRSA isolates showed an overall target attainment of 85.6% for a 1-log(10) CFU/g kill and 79.7% for a 2-log(10) CFU/g kill. It is important to derive exposure targets in preclinical-infection models of the infection site so that these targets can be explored in clinical trials in order to optimize the probability of a good clinical outcome.

  4. Identifying optimal targets of network attack by belief propagation

    CERN Document Server

    Mugisha, Salomon

    2016-01-01

    For a network formed by nodes and undirected links between pairs of nodes, the network optimal attack problem aims at deleting a minimum number of target nodes to break the network down into many small components. This problem is intrinsically related to the feedback vertex set problem that was successfully tackled by spin glass theory and an associated belief propagation-guided decimation (BPD) algorithm [H.-J. Zhou, Eur.~Phys.~J.~B 86 (2013), 455]. In the present work we apply a slightly adjusted version of the BPD algorithm to the network optimal attack problem, and demonstrate that it has much better performance than a recently proposed Collective Information algorithm [F. Morone and H. A. Makse, Nature 524 (2015), 63--68] for different types of random networks and real-world network instances. The BPD-guided attack scheme often induces an abrupt collapse of the whole network, which may make it very difficult to defend.

  5. Identifying optimal targets of network attack by belief propagation

    Science.gov (United States)

    Mugisha, Salomon; Zhou, Hai-Jun

    2016-07-01

    For a network formed by nodes and undirected links between pairs of nodes, the network optimal attack problem aims at deleting a minimum number of target nodes to break the network down into many small components. This problem is intrinsically related to the feedback vertex set problem that was successfully tackled by spin-glass theory and an associated belief propagation-guided decimation (BPD) algorithm [Zhou, Eur. Phys. J. B 86, 455 (2013), 10.1140/epjb/e2013-40690-1]. In the present work we apply the BPD algorithm (which has approximately linear time complexity) to the network optimal attack problem and demonstrate that it has much better performance than a recently proposed collective information algorithm [Morone and Makse, Nature 524, 65 (2015), 10.1038/nature14604] for different types of random networks and real-world network instances. The BPD-guided attack scheme often induces an abrupt collapse of the whole network, which may make it very difficult to defend.

  6. Uveal Melanoma: Identifying Immunological and Chemotherapeutic Targets to Treat Metastases.

    Science.gov (United States)

    Jager, Martine J; Dogrusöz, Mehmet; Woodman, Scott E

    2017-01-01

    Uveal melanoma is an intraocular malignancy that, depending on its size and genetic make-up, may lead to metastases in up to 50% of cases. Currently, no therapy has been proven to improve survival. However, new therapies exploiting immune responses against metastases are being developed. The primary tumor is well characterized: tumors at high risk of developing metastases often contain macrophages and lymphocytes. However, these lymphocytes are often regulatory T cells that may suppress immune response. Currently, immune checkpoint inhibitors have shown marked efficacy in multiple cancers (eg, cutaneous melanoma) but do not yet improve survival in uveal melanoma patients. More knowledge needs to be acquired regarding the function of T cells in uveal melanoma. Other therapeutic options are related to the biochemical pathways. Targeting the RAF-MEK-ERK pathway with small molecule MEK inhibitors abrogates the growth of UM cells harboring GNAQ/GNA11 Q209 mutations, suggesting that these aberrant G-protein oncogenes mediate, at least in part, their effect through this hallmark proliferation pathway. Other pathways are also implicated, such as those involving c-Jun and YAP. Further studies may show how interference in the different pathways may affect survival. Copyright 2017 Asia-Pacific Academy of Ophthalmology.

  7. Targeting the Fanconi Anemia Pathway to Identify Tailored Anticancer Therapeutics

    Directory of Open Access Journals (Sweden)

    Chelsea Jenkins

    2012-01-01

    Full Text Available The Fanconi Anemia (FA pathway consists of proteins involved in repairing DNA damage, including interstrand cross-links (ICLs. The pathway contains an upstream multiprotein core complex that mediates the monoubiquitylation of the FANCD2 and FANCI heterodimer, and a downstream pathway that converges with a larger network of proteins with roles in homologous recombination and other DNA repair pathways. Selective killing of cancer cells with an intact FA pathway but deficient in certain other DNA repair pathways is an emerging approach to tailored cancer therapy. Inhibiting the FA pathway becomes selectively lethal when certain repair genes are defective, such as the checkpoint kinase ATM. Inhibiting the FA pathway in ATM deficient cells can be achieved with small molecule inhibitors, suggesting that new cancer therapeutics could be developed by identifying FA pathway inhibitors to treat cancers that contain defects that are synthetic lethal with FA.

  8. Complementary Approaches to Existing Target Based Drug Discovery for Identifying Novel Drug Targets

    Directory of Open Access Journals (Sweden)

    Suhas Vasaikar

    2016-11-01

    Full Text Available In the past decade, it was observed that the relationship between the emerging New Molecular Entities and the quantum of R&D investment has not been favorable. There might be numerous reasons but few studies stress the introduction of target based drug discovery approach as one of the factors. Although a number of drugs have been developed with an emphasis on a single protein target, yet identification of valid target is complex. The approach focuses on an in vitro single target, which overlooks the complexity of cell and makes process of validation drug targets uncertain. Thus, it is imperative to search for alternatives rather than looking at success stories of target-based drug discovery. It would be beneficial if the drugs were developed to target multiple components. New approaches like reverse engineering and translational research need to take into account both system and target-based approach. This review evaluates the strengths and limitations of known drug discovery approaches and proposes alternative approaches for increasing efficiency against treatment.

  9. Determination of chloramphenicol residues in commercial chicken eggs in the Federal Capital Territory, Abuja, Nigeria.

    Science.gov (United States)

    Mbodi, Felix E; Nguku, P; Okolocha, E; Kabir, J

    2014-01-01

    The use of antibiotics in poultry can result in residues in eggs. The joint FAO/WHO committee recommended banning the use of chloramphenicol (CAP) in food animals due to its public health hazards of aplastic anaemia, leukaemia, allergy, antibacterial resistance and carcinogenicity. This paper determines the prevalence of CAP residues in chicken eggs and assesses the usage and awareness of its ban amongst poultry farmers in the Federal Capital Territory (FCT), Abuja, Nigeria. A cross-sectional survey of registered poultry farmers in FCT was conducted using questionnaires to determine CAP administration in poultry and awareness of its ban. Pooled egg samples were collected from each poultry farm surveyed and from randomly sampled government-owned markets in FCT. Source of eggs by state were identified by the marketer at the time of collection. Samples were analysed using an enzyme-linked immunosorbent assay (ELISA) technique for the presence of CAP, and prevalence was determined. Of 288 total pooled samples collected, 257 (89.2%) were from the markets and 31 (10.8%) were from poultry farms. A total of 20 (7%) pooled egg samples tested CAP-positive; market eggs originated from 15 (41%) states of the country. Of the market eggs, 16 (6.2%) pooled samples tested positive. Of eggs from poultry farms, four (12.9%) tested positive. Mean CAP concentrations in the positive samples ranged from 0.49 to 1.17 µg kg(-1) (parts per billion). CAP use amongst poultry farmers in FCT was 75.5%; awareness of the CAP ban was 26.3%. Though 66% of veterinarians were unaware of a CAP ban, they were more likely to be aware than other poultry farmers (odds ratio (OR) = 1.4). Farm managers who use CAP were more likely to be aware of CAP ban than the farm managers not using CAP (OR = 5.5; p = 0.04). Establishing a drug residue surveillance and control program and enforcement of CAP legislation/regulation is needful to educate and prohibit the widespread CAP use amongst Nigerian poultry farmers.

  10. Whole report uncovers correctly identified but incorrectly placed target information under visual crowding.

    Science.gov (United States)

    Zhang, Jun-Yun; Zhang, Gong-Liang; Liu, Lei; Yu, Cong

    2012-07-10

    Multiletter identification studies often find correctly identified letters being reported in wrong positions. However, how position uncertainty impacts crowding in peripheral vision is not fully understood. The observation of a flanker being reported as the central target cannot be taken as unequivocal evidence for position misperception because the observers could be biased to report a more identifiable flanker when failing to identify the central target. In addition, it has never been reported whether a correctly identified central target can be perceived at a flanker position under crowding. Empirical investigation into this possibility holds the key to demonstrating letter-level position uncertainty in crowding, because the position errors of the least identifiable central target cannot be attributed to response bias. We asked normally-sighted observers to report either the central target of a trigram (partial report) or all three characters (whole report). The results showed that, for radially arranged trigrams, the rate of reporting the central target regardless of the reported position in the whole report was significantly higher than the partial report rate, and the extra target reports mostly ended up in flanker positions. Error analysis indicated that target-flanker position swapping and misalignment (lateral shift of the target and one flanker) underlay this target misplacement. Our results thus establish target misplacement as a source of crowding errors and ascertain the role of letter-level position uncertainty in crowding.

  11. Adherence to vector preventive measures decrease cases of acute Dengue among Abuja residents, Nigeria

    Directory of Open Access Journals (Sweden)

    Idris Abdullahi Nasir

    2017-07-01

    Full Text Available Background: Nigeria is one of the dengue hyper-endemic nations. This study investigated the level of knowledge about dengue and vector preventive practices and their impacts on acute dengue among febrile patients at Abuja, Nigeria. Materials and methods: This cross-sectional survey was conducted on febrile patients attending University of Abuja Teaching Hospital, Abuja, Nigeria. Blood samples were individually collected from 171 febrile patients residing at Gwagwalada suburb. Interviewer administered questionnaires were used to assess subjects’ knowledge about preventive measures against vector breeding and bites. Blood samples were tested for dengue virus Nonstructural glycoprotein-1antigen using enzyme linked Immunosorbent assay (ELISA. Results: Fifteen out of 171 febrile persons (8.8% were Dengue NS1 positive. Sixty percent of the subjects do not know about dengue, while 33% knew about dengue virus infection through television/ radio programs, 5% through healthcare professionals and 2% from friends/families. Those who persistently use indoor residual spraying and long sleeves/trousers during daytime had less cases of DENV NS1 than those who do not. There was statistical association between DENV NS1 and residence in proximity to waste dumpsites (P<0.0001 and frequent use of long sleeve clothing and trousers (P=0.005. However, there was no statistical association between DENV NS1 antigenemia and persistent use of indoor residual spraying and presence of in-door water containers (P>0.05. Conclusions: Findings from this study imply that proper education and adherence to preventive measures minimize people from being susceptible to Dengue virus infections.

  12. Systematic analysis of public domain compound potency data identifies selective molecular scaffolds across druggable target families.

    Science.gov (United States)

    Hu, Ye; Wassermann, Anne Mai; Lounkine, Eugen; Bajorath, Jürgen

    2010-01-28

    Molecular scaffolds that yield target family-selective compounds are of high interest in pharmaceutical research. There continues to be considerable debate in the field as to whether chemotypes with a priori selectivity for given target families and/or targets exist and how they might be identified. What do currently available data tell us? We present a systematic and comprehensive selectivity-centric analysis of public domain target-ligand interactions. More than 200 molecular scaffolds are identified in currently available active compounds that are selective for established target families. A subset of these scaffolds is found to produce compounds with high selectivity for individual targets among closely related ones. These scaffolds are currently underrepresented in approved drugs.

  13. Identifying the Right Disease Targets to Develop Better Drugs, Faster | NIH MedlinePlus the Magazine

    Science.gov (United States)

    ... JavaScript on. Identifying the Right Disease Targets to Develop Better Drugs, Faster Past Issues / Spring 2014 Table ... a-dozen pharmaceutical companies are now racing to develop drugs that lower cholesterol by blocking PCSK9. Are ...

  14. The Use of Social Networking among Senior Secondary School Students in Abuja Municipal Area of Federal Capital Territory, Nigeria

    Science.gov (United States)

    Ali, F. A. Farah; Aliyu, Umar Yanda

    2015-01-01

    The present study examined the use of social networking among senior secondary school students in Abuja Municipal Area Council of FCT. The study employed quantitative method for data collection involving questionnaire administration. Fifteen questions with Likert model and ten yes/no responses in a questionnaire were personally administered to 400…

  15. Principals' Personal Variables and Information and Communication Technology Utilization in Federal Capital Territory Senior Secondary Schools, Abuja, Nigeria

    Science.gov (United States)

    Ogunshola, Roseline Folashade; Adeniyi, Abiodun

    2017-01-01

    The study investigated principals' personal variables and information and communication technology utilization in Federal Capital Territory (FCT) senior secondary schools, Abuja, Nigeria. The study adopted the correlational research design. The study used a sample of 94 senior secondary schools (including public and private) in FCT. Stratified…

  16. A Riboproteomic Platform to Identify Novel Targets for Prostate Cancer Therapy

    Science.gov (United States)

    2016-12-01

    AWARD NUMBER: W81XWH-14-1-0590 TITLE: A Riboproteomic Platform to Identify Novel Targets for Prostate Cancer Therapy PRINCIPAL INVESTIGATOR...2014 - 28 Sep 2016 4. TITLE AND SUBTITLE A Riboproteomic Platform to Identify Novel Targets for Prostate Cancer Therapy 5a. CONTRACT NUMBER 5b...all proteins that constitute the riboproteome, to evaluate which of these proteins are altered between different prostate cancer cell lines and types

  17. Patent medicine vendors, community pharmacists and STI management in Abuja, Nigeria.

    Science.gov (United States)

    Okonkwo, A D; Okonkwo, U P

    2010-09-01

    Increasingly, literature indicates that Patent Medicine Vendors (PMVs) and Community Pharmacists (CPs) provide sexual reproductive health services and products to their young patrons. This study explored the validity of literature claims, principally from CPs and PMVs perspective in Abuja, Nigeria. Participants were recruited with convenience sampling based on their willingness to participate in the study and our judgement of their professional competence. They were administered a semi-structured questionnaire, which was modelled after McCracken's long interview. We empirically assessed the validity of CPs and PMVs opinions with an exit interview of seven consenting patrons. Interviews were audio taped, transcribed verbatim and subjected to iterative thematic analysis. Participants' accounts and our observations indicate that PMVs and CPs serve young people's sexual reproductive healthcare needs in Abuja. CPs and PMVs provide young people with a seamless and non-judgemental access to contraceptives, sexual health advice and post-sexual risk exposure care. The study corroborates literature claims that CPs and PMVs provide sexual reproductive health advice, services and products to young people. However, participants contend that the current pharmacy practice laws in Nigeria constrain the scope and quality of services that young unmarried people require. Because it is unlikely that Nigeria will reinvigorate her primary healthcare system soon, we call for the formal co-option of CPs and PMVs into the sexual reproductive health management system to standardize and improve services.

  18. Sixth Africa malaria day in 2006: how far have we come after the Abuja Declaration?

    Directory of Open Access Journals (Sweden)

    Wanga Charles L

    2006-11-01

    Full Text Available Abstract Each year on the 25th April Africa and the rest of the world commemorate Africa Malaria Day as was agreed upon at the African Summit on Roll Back Malaria held in Abuja, Nigeria on 25th April 2000. The summit also called upon the United Nations to declare the period 2001–2010 a decade for malaria. The 1st Africa Malaria Day was commemorated with the theme "Communities Play a Central Role in Tackling Malaria". The 6th Africa Malaria Day was observed in 2006 with the theme "Get Your ACT Together" and the slogan "Universal Access to Effective Malaria Treatment is a Human Right". This article by the Secretariat of the Multilateral Initiative on Malaria (MIM was also part of the commemorations for the day. MIM was founded in 1997 as an alliance of institutions and individuals concerned with the malaria problem, and aiming at maximizing the impact of scientific research on malaria through strengthening African research capacity and coordinated global collaboration. The MIM Secretariat has been hosted in rotation by the founding institutions, and is being hosted for the first time in Africa by the African Malaria Network Trust (AMANET in Dar es Salaam, Tanzania. This article reviews the malaria situation in Africa six years after the Abuja Declaration, highlighting the disease burden trends, failures, achievements, challenges, and the way forward.

  19. A series of case studies: practical methodology for identifying antinociceptive multi-target drugs.

    Science.gov (United States)

    Pang, Min-Hee; Kim, Yuntae; Jung, Kyung Woon; Cho, Sunyoung; Lee, Doo H

    2012-05-01

    Since the introduction of drug discovery based on single targets, the number of newly developed drugs has steadily declined, and the reliablility of the current drug-discovery paradigm has been unceasingly questioned. As an alternative, an emerging approach pursuing multi-targeting drugs has arisen to reflect multifactorial diseases caused by the complex networks of various mechanisms. The purpose of this paper is to review multi-target drugs and introduce our progress in establishing a practical methodology for identifying antinociceptive multi-target drugs. We have adopted a system of ex vivo efficacy screening using long-term potentiation in rat spinal cord as a surrogate biomarker for neuropathic pain. A bait-target approach is also adopted to lure an unknown target combination that induces synergistic mechanisms.

  20. Identifying master regulators of cancer and their downstream targets by integrating genomic and epigenomic features.

    Science.gov (United States)

    Gevaert, Olivier; Plevritis, Sylvia

    2013-01-01

    Vast amounts of molecular data characterizing the genome, epigenome and transcriptome are becoming available for a variety of cancers. The current challenge is to integrate these diverse layers of molecular biology information to create a more comprehensive view of key biological processes underlying cancer. We developed a biocomputational algorithm that integrates copy number, DNA methylation, and gene expression data to study master regulators of cancer and identify their targets. Our algorithm starts by generating a list of candidate driver genes based on the rationale that genes that are driven by multiple genomic events in a subset of samples are unlikely to be randomly deregulated. We then select the master regulators from the candidate driver and identify their targets by inferring the underlying regulatory network of gene expression. We applied our biocomputational algorithm to identify master regulators and their targets in glioblastoma multiforme (GBM) and serous ovarian cancer. Our results suggest that the expression of candidate drivers is more likely to be influenced by copy number variations than DNA methylation. Next, we selected the master regulators and identified their downstream targets using module networks analysis. As a proof-of-concept, we show that the GBM and ovarian cancer module networks recapitulate known processes in these cancers. In addition, we identify master regulators that have not been previously reported and suggest their likely role. In summary, focusing on genes whose expression can be explained by their genomic and epigenomic aberrations is a promising strategy to identify master regulators of cancer.

  1. Yeast-based assay identifies novel Shh/Gli target genes in vertebrate development

    Directory of Open Access Journals (Sweden)

    Milla Luis A

    2012-01-01

    Full Text Available Abstract Background The increasing number of developmental events and molecular mechanisms associated with the Hedgehog (Hh pathway from Drosophila to vertebrates, suggest that gene regulation is crucial for diverse cellular responses, including target genes not yet described. Although several high-throughput, genome-wide approaches have yielded information at the genomic, transcriptional and proteomic levels, the specificity of Gli binding sites related to direct target gene activation still remain elusive. This study aims to identify novel putative targets of Gli transcription factors through a protein-DNA binding assay using yeast, and validating a subset of targets both in-vitro and in-vivo. Testing in different Hh/Gli gain- and loss-of-function scenarios we here identified known (e.g., ptc1 and novel Hh-regulated genes in zebrafish embryos. Results The combined yeast-based screening and MEME/MAST analysis were able to predict Gli transcription factor binding sites, and position mapping of these sequences upstream or in the first intron of promoters served to identify new putative target genes of Gli regulation. These candidates were validated by qPCR in combination with either the pharmacological Hh/Gli antagonist cyc or the agonist pur in Hh-responsive C3H10T1/2 cells. We also used small-hairpin RNAs against Gli proteins to evaluate targets and confirm specific Gli regulation their expression. Taking advantage of mutants that have been identified affecting different components of the Hh/Gli signaling system in the zebrafish model, we further analyzed specific novel candidates. Studying Hh function with pharmacological inhibition or activation complemented these genetic loss-of-function approaches. We provide evidence that in zebrafish embryos, Hh signaling regulates sfrp2, neo1, and c-myc expression in-vivo. Conclusion A recently described yeast-based screening allowed us to identify new Hh/Gli target genes, functionally important in

  2. Prevalence of vulvovaginal candidiasis among nonpregnant women attending a tertiary health care facility in Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Emeribe AU

    2015-06-01

    Full Text Available Anthony Uchenna Emeribe,1 Idris Abdullahi Nasir,2 Justus Onyia,2 Alinwachukwu Loveth Ifunanya31Department of Medical Laboratory Science, University of Calabar, Calabar, Cross River State, Nigeria; 2Department of Medical Microbiology, University of Abuja Teaching Hospital, Gwagwalada, Federal Capital Territory, Nigeria; 3Department of Medical Laboratory, School of Health Technology, Tsafe, Zamfara State, NigeriaBackground: Candida spp. are normal flora of the vagina that eventually become pathogenic under some prevailing conditions, and thus present as a common etiology of vulvovaginitis. When prompt recognition and laboratory confirmation is not achieved, this could lead to devastating genital discomfort and a major reason for frequent hospital visits.Aims: This was a cross-sectional prospective study that aimed to determine the prevalence and some associated risk factors of vulvovaginal candidiasis (VVC among nonpregnant women attending University of Abuja Teaching Hospital, Gwagwalada.Subjects and methods: A pair of high vaginal swab and endocervical swab samples was collected from each of 200 individual participating subjects. They were separately inoculated on Sabouraud's dextrose agar and incubated aerobically at 33°C for 48 hours. Ten percent KOH wet mount and Gram staining was done on swabs and colonies, respectively. Structured questionnaires were used to obtain sociodemographic and clinical data.Results: Of the 200 participating subjects, the prevalence of Candida albicans was 6.5% and that of non-albicans candidiasis 7.5%. Candidiasis was observed mostly among the 20- to 30-year age-group. All subjects with Candida-positive culture had been on antibacterial therapy prior to participating in this study – 28 (100%. There was a statistical relationship between the prevalence of VVC with previous antibacterial therapy (P<0.05, but not with age or other prevailing health conditions studied (P>0.05.Conclusion: The outcome of this study

  3. Flux variability scanning based on enforced objective flux for identifying gene amplification targets

    Directory of Open Access Journals (Sweden)

    Park Jong

    2012-08-01

    Full Text Available Abstract Background In order to reduce time and efforts to develop microbial strains with better capability of producing desired bioproducts, genome-scale metabolic simulations have proven useful in identifying gene knockout and amplification targets. Constraints-based flux analysis has successfully been employed for such simulation, but is limited in its ability to properly describe the complex nature of biological systems. Gene knockout simulations are relatively straightforward to implement, simply by constraining the flux values of the target reaction to zero, but the identification of reliable gene amplification targets is rather difficult. Here, we report a new algorithm which incorporates physiological data into a model to improve the model’s prediction capabilities and to capitalize on the relationships between genes and metabolic fluxes. Results We developed an algorithm, flux variability scanning based on enforced objective flux (FVSEOF with grouping reaction (GR constraints, in an effort to identify gene amplification targets by considering reactions that co-carry flux values based on physiological omics data via “GR constraints”. This method scans changes in the variabilities of metabolic fluxes in response to an artificially enforced objective flux of product formation. The gene amplification targets predicted using this method were validated by comparing the predicted effects with the previous experimental results obtained for the production of shikimic acid and putrescine in Escherichia coli. Moreover, new gene amplification targets for further enhancing putrescine production were validated through experiments involving the overexpression of each identified targeted gene under condition-controlled batch cultivation. Conclusions FVSEOF with GR constraints allows identification of gene amplification targets for metabolic engineering of microbial strains in order to enhance the production of desired bioproducts. The algorithm

  4. Hypospadias repair and outcome in Abuja, Nigeria: A 5-year single-centre experience

    Science.gov (United States)

    Aisuodionoe-Shadrach, Oseremen Inokhoife; Atim, Terkaa; Eniola, Bolarinwa Sefiu; Ohemu, Alexander Akogwu

    2015-01-01

    Background: To determine the outcome of hypospadias repair in children. Patients and Methods: This was a retrospective study of all patients with hypospadias managed at the University of Abuja Teaching Hospital, Abuja, Nigeria from January 2009 to December 2013. Results: Twenty-four cases of hypospadias had corrective surgery during the 5-year period under review. Seventy-five percent of the patients (n = 18) were seen after the 1st year of life. There were two peaks of ages at corrective repair; 45.8% between age 1 and 3 years and 29.1% between age 5 and 10 years. The average age at time of surgery was 44.9 months. Distal hypospadias were more common (58.4%), followed by glanular (20.8%) and proximal (20.8%) hypospadias. Associated anomalies included chordee, maldescended testicles and inguinal hernia in 20.8%, 4.1% and 8.3% cases, respectively. Operative techniques were single-stage procedures in 79.1% of patients consisting of simple circumcision in two cases (10.5%), Mathieu's peri-meatal based flap in four cases (21%), meatal advancement and glanuloplasty incorporated in three cases (16%) and Snodgrass tubularised incised urethral plate tubularised incised plate in 10 cases (52.5%). The remaining 20.9% (n = 5) had multi-staged procedures. The most common post-operative complications were urethrocutenous fistula in nine patients (33.3%) and metal stenosis in 3 patients (12.5%). Conclusions: Our results show that hypospadia repair is froth with attendant high complications in our setting. PMID:25659549

  5. Targeted resequencing in epileptic encephalopathies identifies de novo mutations in CHD2 and SYNGAP1

    DEFF Research Database (Denmark)

    Carvill, Gemma L; Heavin, Sinéad B; Yendle, Simone C

    2013-01-01

    Epileptic encephalopathies are a devastating group of epilepsies with poor prognosis, of which the majority are of unknown etiology. We perform targeted massively parallel resequencing of 19 known and 46 candidate genes for epileptic encephalopathy in 500 affected individuals (cases) to identify ...

  6. Identifying target groups for the prevention of depression in early adolescence : The TRAILS study

    NARCIS (Netherlands)

    Monshouwer, K.; Smit, F.; Ruiter, M.; Ormel, Johan; Verhulst, F.; Vollebergh, W.; Oldehinkel, T.

    Background: Depression in adolescence is associated with long-term adverse consequences. The aim of the present study is to identify target groups at increased risk of developing depression in early adolescence, such that prevention is associated with the largest health benefit at population-level

  7. Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

    LENUS (Irish Health Repository)

    Toomey, David

    2009-01-01

    BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins\\/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and\\/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY\\/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i) homologous to previously crystallized proteins or (ii) targets of known drugs, but are (iii) not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS\\/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under \\'change-of-application\\' patents.

  8. Genomes2Drugs: identifies target proteins and lead drugs from proteome data.

    Directory of Open Access Journals (Sweden)

    David Toomey

    Full Text Available BACKGROUND: Genome sequencing and bioinformatics have provided the full hypothetical proteome of many pathogenic organisms. Advances in microarray and mass spectrometry have also yielded large output datasets of possible target proteins/genes. However, the challenge remains to identify new targets for drug discovery from this wealth of information. Further analysis includes bioinformatics and/or molecular biology tools to validate the findings. This is time consuming and expensive, and could fail to yield novel drugs if protein purification and crystallography is impossible. To pre-empt this, a researcher may want to rapidly filter the output datasets for proteins that show good homology to proteins that have already been structurally characterised or proteins that are already targets for known drugs. Critically, those researchers developing novel antibiotics need to select out the proteins that show close homology to any human proteins, as future inhibitors are likely to cross-react with the host protein, causing off-target toxicity effects later in clinical trials. METHODOLOGY/PRINCIPAL FINDINGS: To solve many of these issues, we have developed a free online resource called Genomes2Drugs which ranks sequences to identify proteins that are (i homologous to previously crystallized proteins or (ii targets of known drugs, but are (iii not homologous to human proteins. When tested using the Plasmodium falciparum malarial genome the program correctly enriched the ranked list of proteins with known drug target proteins. CONCLUSIONS/SIGNIFICANCE: Genomes2Drugs rapidly identifies proteins that are likely to succeed in drug discovery pipelines. This free online resource helps in the identification of potential drug targets. Importantly, the program further highlights proteins that are likely to be inhibited by FDA-approved drugs. These drugs can then be rapidly moved into Phase IV clinical studies under 'change-of-application' patents.

  9. Identifying gene targets for the metabolic engineering of lycopene biosynthesis in Escherichia coli.

    Science.gov (United States)

    Alper, Hal; Jin, Yong-Su; Moxley, J F; Stephanopoulos, G

    2005-05-01

    The identification of genetic targets that are effective in bringing about a desired phenotype change is still an open problem. While random gene knockouts have yielded improved strains in certain cases, it is also important to seek the guidance of cell-wide stoichiometric constraints in identifying promising gene knockout targets. To investigate these issues, we undertook a genome-wide stoichiometric flux balance analysis as an aid in discovering putative genes impacting network properties and cellular phenotype. Specifically, we calculated metabolic fluxes such as to optimize growth and then scanned the genome for single and multiple gene knockouts that yield improved product yield while maintaining acceptable overall growth rate. For the particular case of lycopene biosynthesis in Escherichia coli, we identified such targets that we subsequently tested experimentally by constructing the corresponding single, double and triple gene knockouts. While such strains are suggested (by the stoichiometric calculations) to increase precursor availability, this beneficial effect may be further impacted by kinetic and regulatory effects not captured by the stoichiometric model. For the case of lycopene biosynthesis, the so identified knockout targets yielded a triple knockout construct that exhibited a nearly 40% increase over an engineered, high producing parental strain.

  10. Candidate cancer-targeting agents identified by expression-profiling arrays

    Directory of Open Access Journals (Sweden)

    Termglinchan V

    2013-04-01

    Full Text Available Vittavat Termglinchan,1 Wachiraporn Wanichnopparat,1 Kulachanya Suwanwongse,1 Chunhakarn Teeyapant,1 Kanticha Chatpermporn,1 Kanchana Leerunyakul,1 Khwanruthai Chuadpia,1 Onpailin Sirimaneethum,1 Parinya Wijitworawong,1 Wattanakitch Mutirangura,1 Chatchawit Aporntewan,2 Chanida Vinayanuwattikun,3 Apiwat Mutirangura4 1Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand; 2Department of Mathematics and Computer Science, Faculty of Science, Chulalongkorn University, Bangkok, Thailand; 3Division of Medical Oncology, Department of Medicine, Faculty of Medicine, Chulalongkorn University and The King Chulalongkorn Memorial Hospital, Bangkok, Thailand; 4Center of Excellence in Molecular Genetics of Cancer and Human Diseases, Department of Anatomy, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand Background: One particularly promising component of personalized medicine in cancer treatment is targeted therapy, which aims to maximize therapeutic efficacy while minimizing toxicity. However, the number of approved targeted agents remains limited. Expression microarray data for different types of cancer are resources to identify genes that were upregulated. The genes are candidate targets for cancer-targeting agents for future anticancer research and targeted treatments. Methods and findings: The gene expression profiles of 48 types of cancer from 2,141 microarrays reported in the Gene Expression Omnibus were analyzed. These data were organized into 78 experimental groups, on which we performed comprehensive analyses using two-tailed Student's t-tests with significance set at P < 0.01 to identify genes that were upregulated compared with normal cells in each cancer type. The resulting list of significantly upregulated genes was cross-referenced with three categories of protein inhibitor targets, categorized by inhibitor type ('Targets of US Food and Drug Administration (FDA-approved anticancer drugs', 'Targets of FDA

  11. Synthetic lethal RNAi screening identifies sensitizing targets for gemcitabine therapy in pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Azorsa David O

    2009-06-01

    Full Text Available Abstract Background Pancreatic cancer retains a poor prognosis among the gastrointestinal cancers. It affects 230,000 individuals worldwide, has a very high mortality rate, and remains one of the most challenging malignancies to treat successfully. Treatment with gemcitabine, the most widely used chemotherapeutic against pancreatic cancer, is not curative and resistance may occur. Combinations of gemcitabine with other chemotherapeutic drugs or biological agents have resulted in limited improvement. Methods In order to improve gemcitabine response in pancreatic cancer cells, we utilized a synthetic lethal RNAi screen targeting 572 known kinases to identify genes that when silenced would sensitize pancreatic cancer cells to gemcitabine. Results Results from the RNAi screens identified several genes that, when silenced, potentiated the growth inhibitory effects of gemcitabine in pancreatic cancer cells. The greatest potentiation was shown by siRNA targeting checkpoint kinase 1 (CHK1. Validation of the screening results was performed in MIA PaCa-2 and BxPC3 pancreatic cancer cells by examining the dose response of gemcitabine treatment in the presence of either CHK1 or CHK2 siRNA. These results showed a three to ten-fold decrease in the EC50 for CHK1 siRNA-treated cells versus control siRNA-treated cells while treatment with CHK2 siRNA resulted in no change compared to controls. CHK1 was further targeted with specific small molecule inhibitors SB 218078 and PD 407824 in combination with gemcitabine. Results showed that treatment of MIA PaCa-2 cells with either of the CHK1 inhibitors SB 218078 or PD 407824 led to sensitization of the pancreatic cancer cells to gemcitabine. Conclusion These findings demonstrate the effectiveness of synthetic lethal RNAi screening as a tool for identifying sensitizing targets to chemotherapeutic agents. These results also indicate that CHK1 could serve as a putative therapeutic target for sensitizing pancreatic cancer

  12. Evaluation of approaches to identify the targets of cellular immunity on a proteome-wide scale.

    Directory of Open Access Journals (Sweden)

    Fernanda C Cardoso

    Full Text Available BACKGROUND: Vaccine development against malaria and other complex diseases remains a challenge for the scientific community. The recent elucidation of the genome, proteome and transcriptome of many of these complex pathogens provides the basis for rational vaccine design by identifying, on a proteome-wide scale, novel target antigens that are recognized by T cells and antibodies from exposed individuals. However, there is currently no algorithm to effectively identify important target antigens from genome sequence data; this is especially challenging for T cell targets. Furthermore, for some of these pathogens, such as Plasmodium, protein expression using conventional platforms has been problematic but cell-free in vitro transcription translation (IVTT strategies have recently proved successful. Herein, we report a novel approach for proteome-wide scale identification of the antigenic targets of T cell responses using IVTT products. PRINCIPAL FINDINGS: We conducted a series of in vitro and in vivo experiments using IVTT proteins either unpurified, absorbed to carboxylated polybeads, or affinity purified through nickel resin or magnetic beads. In vitro studies in humans using CMV, EBV, and Influenza A virus proteins showed antigen-specific cytokine production in ELIspot and Cytometric Bead Array assays with cells stimulated with purified or unpurified IVTT antigens. In vitro and in vivo studies in mice immunized with the Plasmodium yoelii circumsporozoite DNA vaccine with or without IVTT protein boost showed antigen-specific cytokine production using purified IVTT antigens only. Overall, the nickel resin method of IVTT antigen purification proved optimal in both human and murine systems. CONCLUSIONS: This work provides proof of concept for the potential of high-throughput approaches to identify T cell targets of complex parasitic, viral or bacterial pathogens from genomic sequence data, for rational vaccine development against emerging and re

  13. Combined Proteomic-Molecular Epidemiology Approach to Identify Precision Targets in Brain Cancer.

    Science.gov (United States)

    Mostovenko, Ekaterina; Liu, Yanhong; Amirian, E Susan; Tsavachidis, Spiridon; Armstrong, Georgina N; Bondy, Melissa L; Nilsson, Carol L

    2017-07-11

    Primary brain tumors are predominantly malignant gliomas. Grade IV astrocytomas (glioblastomas, GBM) are among the most deadly of all tumors; most patients will succumb to their disease within 2 years of diagnosis despite standard of care. The grim outlook for brain tumor patients indicates that novel precision therapeutic targets must be identified. Our hypothesis is that the cancer proteomes of glioma tumors may contain protein variants that are linked to the aggressive pathology of the disease. To this end, we devised a novel workflow that combined variant proteomics with molecular epidemiological mining of public cancer data sets to identify 10 previously unrecognized variants linked to the risk of death in low grade glioma or GBM. We hypothesize that a subset of the protein variants may be successfully developed in the future as novel targets for malignant gliomas.

  14. A simple yeast-based strategy to identify host cellular processes targeted by bacterial effector proteins.

    Directory of Open Access Journals (Sweden)

    Eran Bosis

    Full Text Available Bacterial effector proteins, which are delivered into the host cell via the type III secretion system, play a key role in the pathogenicity of gram-negative bacteria by modulating various host cellular processes to the benefit of the pathogen. To identify cellular processes targeted by bacterial effectors, we developed a simple strategy that uses an array of yeast deletion strains fitted into a single 96-well plate. The array is unique in that it was optimized computationally such that despite the small number of deletion strains, it covers the majority of genes in the yeast synthetic lethal interaction network. The deletion strains in the array are screened for hypersensitivity to the expression of a bacterial effector of interest. The hypersensitive deletion strains are then analyzed for their synthetic lethal interactions to identify potential targets of the bacterial effector. We describe the identification, using this approach, of a cellular process targeted by the Xanthomonas campestris type III effector XopE2. Interestingly, we discover that XopE2 affects the yeast cell wall and the endoplasmic reticulum stress response. More generally, the use of a single 96-well plate makes the screening process accessible to any laboratory and facilitates the analysis of a large number of bacterial effectors in a short period of time. It therefore provides a promising platform for studying the functions and cellular targets of bacterial effectors and other virulence proteins.

  15. Integrative omics analysis of rheumatoid arthritis identifies non-obvious therapeutic targets.

    Directory of Open Access Journals (Sweden)

    John W Whitaker

    Full Text Available Identifying novel therapeutic targets for the treatment of disease is challenging. To this end, we developed a genome-wide approach of candidate gene prioritization. We independently collocated sets of genes that were implicated in rheumatoid arthritis (RA pathogenicity through three genome-wide assays: (i genome-wide association studies (GWAS, (ii differentially expression in RA fibroblast-like synoviocytes (FLS, and (iii differentially methylation in RA FLS. Integrated analysis of these complementary data sets identified a significant enrichment of multi-evidence genes (MEGs within pathways relating to RA pathogenicity. One MEG is Engulfment and Cell Motility Protein-1 (ELMO1, a gene not previously considered as a therapeutic target in RA FLS. We demonstrated in RA FLS that ELMO1 is: (i expressed, (ii promotes cell migration and invasion, and (iii regulates Rac1 activity. Thus, we created links between ELMO1 and RA pathogenicity, which in turn validates ELMO1 as a potential RA therapeutic target. This study illustrated the power of MEG-based approaches for therapeutic target identification.

  16. A target based approach identifies genomic predictors of breast cancer patient response to chemotherapy

    Directory of Open Access Journals (Sweden)

    Hallett Robin M

    2012-05-01

    Full Text Available Abstract Background The efficacy of chemotherapy regimens in breast cancer patients is variable and unpredictable. Whether individual patients either achieve long-term remission or suffer recurrence after therapy may be dictated by intrinsic properties of their breast tumors including genetic lesions and consequent aberrant transcriptional programs. Global gene expression profiling provides a powerful tool to identify such tumor-intrinsic transcriptional programs, whose analyses provide insight into the underlying biology of individual patient tumors. For example, multi-gene expression signatures have been identified that can predict the likelihood of disease reccurrence, and thus guide patient prognosis. Whereas such prognostic signatures are being introduced in the clinical setting, similar signatures that predict sensitivity or resistance to chemotherapy are not currently clinically available. Methods We used gene expression profiling to identify genes that were co-expressed with genes whose transcripts encode the protein targets of commonly used chemotherapeutic agents. Results Here, we present target based expression indices that predict breast tumor response to anthracycline and taxane based chemotherapy. Indeed, these signatures were independently predictive of chemotherapy response after adjusting for standard clinic-pathological variables such as age, grade, and estrogen receptor status in a cohort of 488 breast cancer patients treated with adriamycin and taxotere/taxol. Conclusions Importantly, our findings suggest the practicality of developing target based indices that predict response to therapeutics, as well as highlight the possibility of using gene signatures to guide the use of chemotherapy during treatment of breast cancer patients.

  17. Assessment of an electronic wallet system and determinants of cassava farmers’ participation in off-farm activities in Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Alabi Olugbenga Omotayo

    2016-01-01

    Full Text Available This study assessed an electronic wallet (e-wallet system and determinants of cassava farmers’ participation in off-farm activities in Abuja, Nigeria. A sample of 67 cassava farmers was selected through a multi-stage sampling technique from six selected agricultural extension blocks. Primary data were used. Data were collected using a well-structured and well-designed questionnaire. The analytical tools used were descriptive statistics and maximum likelihood estimates using the logistic regression model. The results indicated that an electronic-wallet is an innovative, motivational mobile phone technology, the first in sub-Saharan Africa to distribute mobile phones to farmers which bring fertilizers, improved seeds, financial services, and agricultural information tips to farmers, and this increases yields and output. The electronic wallet system targeted 20 million farmers with an intention to distribute 10 million mobile phones to farmers in 4 years. In 2014, 1,000 agro-dealers and fourteen million five hundred farmers were registered on the electronic wallet system respectively. Also, one million two hundred farmers have also received inputs via the electronic-wallet system. The electronic-wallet system added an estimated 30-40 billion dollars to Nigeria’s Gross Domestic Product in 2014. Furthermore, the result shows that the age of most sampled cassava farmers fell between 36 and 50 years. About 94% of sampled cassava farmers were male. Off-farm income has the potential to improve the income of farmers. These types of activities undertaken by cassava farmers off the farms include: teaching, tailoring, hunting, pottery, blacksmithing, petty trading, etc. Farmers’ age, farming experience and number of off-farm activities were significant variables influencing farmers’ participation in off-farm activities at P ≤ 0.01; P ≤ 0.10; and P ≤ 0.01 respectively. The coefficient of Nagelkerke (R2 value was 0.751.This shows that the

  18. TargetLink, a new method for identifying the endogenous target set of a specific microRNA in intact living cells.

    Science.gov (United States)

    Xu, Yan; Chen, Yan; Li, Daliang; Liu, Qing; Xuan, Zhenyu; Li, Wen-Hong

    2017-02-01

    MicroRNAs are small non-coding RNAs acting as posttranscriptional repressors of gene expression. Identifying mRNA targets of a given miRNA remains an outstanding challenge in the field. We have developed a new experimental approach, TargetLink, that applied locked nucleic acid (LNA) as the affinity probe to enrich target genes of a specific microRNA in intact cells. TargetLink also consists a rigorous and systematic data analysis pipeline to identify target genes by comparing LNA-enriched sequences between experimental and control samples. Using miR-21 as a test microRNA, we identified 12 target genes of miR-21 in a human colorectal cancer cell by this approach. The majority of the identified targets interacted with miR-21 via imperfect seed pairing. Target validation confirmed that miR-21 repressed the expression of the identified targets. The cellular abundance of the identified miR-21 target transcripts varied over a wide range, with some targets expressed at a rather low level, confirming that both abundant and rare transcripts are susceptible to regulation by microRNAs, and that TargetLink is an efficient approach for identifying the target set of a specific microRNA in intact cells. C20orf111, one of the novel targets identified by TargetLink, was found to reside in the nuclear speckle and to be reliably repressed by miR-21 through the interaction at its coding sequence.

  19. In silico mining identifies IGFBP3 as a novel target of methylation in prostate cancer.

    LENUS (Irish Health Repository)

    Perry, A S

    2007-05-21

    Promoter hypermethylation is central in deregulating gene expression in cancer. Identification of novel methylation targets in specific cancers provides a basis for their use as biomarkers of disease occurrence and progression. We developed an in silico strategy to globally identify potential targets of promoter hypermethylation in prostate cancer by screening for 5\\' CpG islands in 631 genes that were reported as downregulated in prostate cancer. A virtual archive of 338 potential targets of methylation was produced. One candidate, IGFBP3, was selected for investigation, along with glutathione-S-transferase pi (GSTP1), a well-known methylation target in prostate cancer. Methylation of IGFBP3 was detected by quantitative methylation-specific PCR in 49\\/79 primary prostate adenocarcinoma and 7\\/14 adjacent preinvasive high-grade prostatic intraepithelial neoplasia, but in only 5\\/37 benign prostatic hyperplasia (P < 0.0001) and in 0\\/39 histologically normal adjacent prostate tissue, which implies that methylation of IGFBP3 may be involved in the early stages of prostate cancer development. Hypermethylation of IGFBP3 was only detected in samples that also demonstrated methylation of GSTP1 and was also correlated with Gleason score > or =7 (P=0.01), indicating that it has potential as a prognostic marker. In addition, pharmacological demethylation induced strong expression of IGFBP3 in LNCaP prostate cancer cells. Our concept of a methylation candidate gene bank was successful in identifying a novel target of frequent hypermethylation in early-stage prostate cancer. Evaluation of further relevant genes could contribute towards a methylation signature of this disease.

  20. Novel modeling of combinatorial miRNA targeting identifies SNP with potential role in bone density.

    Directory of Open Access Journals (Sweden)

    Claudia Coronnello

    Full Text Available MicroRNAs (miRNAs are post-transcriptional regulators that bind to their target mRNAs through base complementarity. Predicting miRNA targets is a challenging task and various studies showed that existing algorithms suffer from high number of false predictions and low to moderate overlap in their predictions. Until recently, very few algorithms considered the dynamic nature of the interactions, including the effect of less specific interactions, the miRNA expression level, and the effect of combinatorial miRNA binding. Addressing these issues can result in a more accurate miRNA:mRNA modeling with many applications, including efficient miRNA-related SNP evaluation. We present a novel thermodynamic model based on the Fermi-Dirac equation that incorporates miRNA expression in the prediction of target occupancy and we show that it improves the performance of two popular single miRNA target finders. Modeling combinatorial miRNA targeting is a natural extension of this model. Two other algorithms show improved prediction efficiency when combinatorial binding models were considered. ComiR (Combinatorial miRNA targeting, a novel algorithm we developed, incorporates the improved predictions of the four target finders into a single probabilistic score using ensemble learning. Combining target scores of multiple miRNAs using ComiR improves predictions over the naïve method for target combination. ComiR scoring scheme can be used for identification of SNPs affecting miRNA binding. As proof of principle, ComiR identified rs17737058 as disruptive to the miR-488-5p:NCOA1 interaction, which we confirmed in vitro. We also found rs17737058 to be significantly associated with decreased bone mineral density (BMD in two independent cohorts indicating that the miR-488-5p/NCOA1 regulatory axis is likely critical in maintaining BMD in women. With increasing availability of comprehensive high-throughput datasets from patients ComiR is expected to become an essential

  1. Transcript profiling of Elf5+/- mammary glands during pregnancy identifies novel targets of Elf5.

    Directory of Open Access Journals (Sweden)

    Renee L Rogers

    Full Text Available BACKGROUND: Elf5, an epithelial specific Ets transcription factor, plays a crucial role in the pregnancy-associated development of the mouse mammary gland. Elf5(-/- embryos do not survive, however the Elf5(+/- mammary gland displays a severe pregnancy-associated developmental defect. While it is known that Elf5 is crucial for correct mammary development and lactation, the molecular mechanisms employed by Elf5 to exert its effects on the mammary gland are largely unknown. PRINCIPAL FINDINGS: Transcript profiling was used to investigate the transcriptional changes that occur as a result of Elf5 haploinsufficiency in the Elf5(+/- mouse model. We show that the development of the mouse Elf5(+/- mammary gland is delayed at a transcriptional and morphological level, due to the delayed increase in Elf5 protein in these glands. We also identify a number of potential Elf5 target genes, including Mucin 4, whose expression, is directly regulated by the binding of Elf5 to an Ets binding site within its promoter. CONCLUSION: We identify novel transcriptional targets of Elf5 and show that Muc4 is a direct target of Elf5, further elucidating the mechanisms through which Elf5 regulates proliferation and differentiation in the mammary gland.

  2. Omen: identifying potential spear-phishing targets before the email is sent.

    Energy Technology Data Exchange (ETDEWEB)

    Wendt, Jeremy Daniel.

    2013-07-01

    We present the results of a two year project focused on a common social engineering attack method called "spear phishing". In a spear phishing attack, the user receives an email with information specifically focused on the user. This email contains either a malware-laced attachment or a link to download the malware that has been disguised as a useful program. Spear phishing attacks have been one of the most effective avenues for attackers to gain initial entry into a target network. This project focused on a proactive approach to spear phishing. To create an effective, user-specific spear phishing email, the attacker must research the intended recipient. We believe that much of the information used by the attacker is provided by the target organization's own external website. Thus when researching potential targets, the attacker leaves signs of his research in the webserver's logs. We created tools and visualizations to improve cybersecurity analysts' abilities to quickly understand a visitor's visit patterns and interests. Given these suspicious visitors and log-parsing tools, analysts can more quickly identify truly suspicious visitors, search for potential spear-phishing targeted users, and improve security around those users before the spear phishing email is sent.

  3. Chemical proteomics approaches for identifying the cellular targets of natural products.

    Science.gov (United States)

    Wright, M H; Sieber, S A

    2016-05-01

    Covering: 2010 up to 2016Deconvoluting the mode of action of natural products and drugs remains one of the biggest challenges in chemistry and biology today. Chemical proteomics is a growing area of chemical biology that seeks to design small molecule probes to understand protein function. In the context of natural products, chemical proteomics can be used to identify the protein binding partners or targets of small molecules in live cells. Here, we highlight recent examples of chemical probes based on natural products and their application for target identification. The review focuses on probes that can be covalently linked to their target proteins (either via intrinsic chemical reactivity or via the introduction of photocrosslinkers), and can be applied "in situ" - in living systems rather than cell lysates. We also focus here on strategies that employ a click reaction, the copper-catalysed azide-alkyne cycloaddition reaction (CuAAC), to allow minimal functionalisation of natural product scaffolds with an alkyne or azide tag. We also discuss 'competitive mode' approaches that screen for natural products that compete with a well-characterised chemical probe for binding to a particular set of protein targets. Fuelled by advances in mass spectrometry instrumentation and bioinformatics, many modern strategies are now embracing quantitative proteomics to help define the true interacting partners of probes, and we highlight the opportunities this rapidly evolving technology provides in chemical proteomics. Finally, some of the limitations and challenges of chemical proteomics approaches are discussed.

  4. A phenotypic assay to identify Chikungunya virus inhibitors targeting the nonstructural protein nsP2.

    Science.gov (United States)

    Lucas-Hourani, Marianne; Lupan, Alexandru; Desprès, Philippe; Thoret, Sylviane; Pamlard, Olivier; Dubois, Joëlle; Guillou, Catherine; Tangy, Frédéric; Vidalain, Pierre-Olivier; Munier-Lehmann, Hélène

    2013-02-01

    Chikungunya virus (CHIKV) is a mosquito-transmitted pathogen responsible for an acute infection of abrupt onset, characterized by high fever, polyarthralgia, myalgia, headaches, chills, and rash. In 2006, CHIKV was responsible for an epidemic outbreak of unprecedented magnitude in the Indian Ocean, stressing the need for therapeutic approaches. Since then, we have acquired a better understanding of CHIKV biology, but we are still missing active molecules against this reemerging pathogen. We recently reported that the nonstructural nsP2 protein of CHIKV induces a transcriptional shutoff that allows the virus to block cellular antiviral response. This was demonstrated using various luciferase-based reporter gene assays, including a trans-reporter system where Gal4 DNA binding domain is fused to Fos transcription factor. Here, we turned this assay into a high-throughput screening system to identify small molecules targeting nsP2-mediated shutoff. Among 3040 molecules tested, we identified one natural compound that partially blocks nsP2 activity and inhibits CHIKV replication in vitro. This proof of concept suggests that similar functional assays could be developed to target other viral proteins mediating a cellular shutoff and identify innovative therapeutic molecules.

  5. Exome sequencing of hepatocellular carcinomas identifies new mutational signatures and potential therapeutic targets

    Science.gov (United States)

    Alexandrov, Ludmil B; Calderaro, Julien; Rebouissou, Sandra; Couchy, Gabrielle; Meiller, Clément; Shinde, Jayendra; Soysouvanh, Frederic; Calatayud, Anna-Line; Pinyol, Roser; Pelletier, Laura; Balabaud, Charles; Laurent, Alexis; Blanc, Jean-Frederic; Mazzaferro, Vincenzo; Calvo, Fabien; Villanueva, Augusto; Nault, Jean-Charles; Bioulac-Sage, Paulette; Stratton, Michael R; Llovet, Josep M; Zucman-Rossi, Jessica

    2015-01-01

    Genomic analyses promise to improve tumor characterization in order to optimize personalized treatment for patients with hepatocellular carcinoma (HCC). Exome sequencing analysis of 243 liver tumors revealed mutational signatures associated with specific risk factors, mainly combined alcohol/tobacco consumption, and aflatoxin B1. We identified 161 putative driver genes associated with 11 recurrent pathways. Associations of mutations defined 3 groups of genes related to risk factors and centered on CTNNB1 (alcohol), TP53 (HBV), and AXIN1. Analyses according to tumor stage progression revealed TERT promoter mutation as an early event whereas FGF3, FGF4, FGF19/CCND1 amplification, TP53 and CDKN2A alterations, appeared at more advanced stages in aggressive tumors. In 28% of the tumors we identified genetic alterations potentially targetable by FDA-approved drugs. In conclusion, we identified risk factor-specific mutational signatures and defined the extensive landscape of altered genes and pathways in HCC which will be useful to design clinical trials for targeted therapy. PMID:25822088

  6. Direct targets of the tomato-ripening regulator RIN identified by transcriptome and chromatin immunoprecipitation analyses.

    Science.gov (United States)

    Fujisawa, Masaki; Shima, Yoko; Higuchi, Naoki; Nakano, Toshitsugu; Koyama, Yoshiyuki; Kasumi, Takafumi; Ito, Yasuhiro

    2012-06-01

    The physiological and biochemical changes in fruit ripening produce key attributes of fruit quality including color, taste, aroma and texture. These changes are driven by the highly regulated and synchronized activation of a huge number of ripening-associated genes. In tomato (Solanum lycopersicum), a typical climacteric fruit, the MADS-box transcription factor RIN is one of the earliest-acting ripening regulators, required for both ethylene-dependent and ethylene-independent pathways. Although we previously identified several direct RIN targets, many additional targets remain unidentified, likely including key ripening-associated genes. Here, we report the identification of novel RIN targets by transcriptome and chromatin immunoprecipitation (ChIP) analyses. Transcriptome comparisons by microarray of wild-type and rin mutant tomatoes identified 342 positively regulated genes and 473 negatively regulated genes by RIN during ripening. Most of the positively regulated genes contained possible RIN-binding (CArG-box) sequences in their promoters. Subsequently, we selected six genes from the positively regulated genes and a ripening regulator gene, CNR, and assayed their promoters by quantitative ChIP-PCR to examine RIN binding. All of the seven genes, which are involved in cell wall modification, aroma and flavor development, pathogen defense and transcriptional regulation during ripening, are targets of RIN, suggesting that RIN may control multiple diverse ripening processes. In particular, RIN directly regulates the expression of the ripening-associated transcription factors, CNR, TDR4 and a GRAS family gene, providing an important clue to elucidate the complicated transcriptional cascade for fruit ripening.

  7. Information Needs and Use of Library Resources by Special Needs Students in Selected Government Schools in Kaduna State and Federal Capital Territory, Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Christiana Alami Atabor

    2015-12-01

    Full Text Available This study is on the information needs and use of library resources by special needs students in selected government schools in Kaduna State and Federal Capital Territory, Abuja, Nigeria. The survey technique was employed in the study. Five schools (i.e., Kaduna State Special Education School; Government Technical College, Malali, Kaduna; Alhudahuda College Zaria; Government Secondary School, Kwali and Government Secondary School, Kuje were purposefully selected out of a total of seven. Data were collected using a structured questionnaire for two respondent groups (i.e., special needs students and teachers/librarians. A total of 5 teachers/librarians and 345 special needs students were selected for the study. The data collected were analyzed using descriptive statistics. The findings of the study show that the special needs students have high needs for information on education, employment opportunities, health matter, and human rights. The major information resources in the libraries are books, magazines, newspapers, and few Braille materials. In general, special needs students have a moderate level of satisfaction with the resources in the libraries. The major issue identified by special needs students is insufficient information resources, especially in Braille. On the part of the teachers/librarians, inadequate funds for the acquisition of information resources and the employment of qualified librarians/teachers are found to be the major challenges faced by the libraries.

  8. Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target

    DEFF Research Database (Denmark)

    Yang, Zhiping; Huang, Yuh-Chin T; Koziel, Henry

    2014-01-01

    To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance......). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host...... survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza....

  9. Skin Prick Test Analysis in Allergic Rhinitis Patients: A Preliminary Study in Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    P. U. Ibekwe

    2016-01-01

    Full Text Available Allergic rhinitis (AR is prevalent in Nigeria, though little information exists on the allergen. We assessed the clinical features of AR patients in our environment based on the allergic rhinitis impact on asthma (ARIA classification. Only patients with positive skin prick test (SPT were recruited. Seventy-four patients participated in the study. AR and asthma comorbidity were observed in 13.5%. The proportion of “sneezers-runners” was higher than “blockers” with significantly more “sneezers-runners” having persistent AR (P=0.007. No relationship was established between these predominant symptoms and the aeroallergens used in this study. Intermittent mild and moderate/severe AR were evident in 13.5% and 31.1%, while persistent mild and moderate/severe were seen in 20.3% and 35.1%, respectively. House dust mites allergen yielded the highest number of positive responses (22.6% followed by tree pollen (16.8%. No relationship was observed between the allergens tested and AR severity. Majority of patients were oligosensitive (33.8% and polysensitive (35.1% and were not significantly associated with AR severity (P=0.07. Most AR patients presenting for treatment in Abuja, Nigeria, had moderate-severe persistent AR and showed similar SPT sensitization pattern with countries having similar climatic conditions. Sensitization patterns were not related to ARIA classification or predominant AR symptoms.

  10. Categorisation Of Clay Deposits In The Federal Capital Territory Of Abuja

    Directory of Open Access Journals (Sweden)

    Manukaji John U.

    2013-03-01

    Full Text Available Clay deposits in the Federal Capital Territory of Abuja were investigated with a view to categorizing them in order to determine their suitability as insulating refractory material. Thesamples were collected from three different locations in the territory, namely Sheda, Abaji and Karimu, and labeled A, B, and C. In order toproject a better representation of the territory, the samples were mined from ten cites each. The mined clay samples from the ten cites were mixed properly and a representative specimen for test from that location was produced using the cone and quartering system as recommended by the American Society of Testing Materials (ASTM. Atomic Absorption Spectrometer(AAS was used to determine the chemical composition, while other established processes were used to determine other insulating properties likeparticle size distribution, specific gravity, bulk density, solid density, water absorption, apparent porosity, permeability to air, refractoriness, thermal shock resistance, modulus of rupture, linear shrinkage and thermal conductivity. The chemical analysis showed that all the samples had high percentages of silica and alumina, thereby classifying them as Alumino-silicates.

  11. Aetiology and presentation of ankylosis of the temporomandibular joint: report of 23 cases from Abuja, Nigeria.

    Science.gov (United States)

    Bello, Seidu Adebayo; Aluko Olokun, Bayo; Olaitan, Abayomi Ademola; Ajike, Sunday O

    2012-01-01

    Several studies have reviewed the management of ankylosis of the temporomandibular joint (TMJ), but only a few focused on the aetiology and clinical features. We retrospectively studied the aetiology and clinical features of patients with ankylosis of the TMJ who presented to the Maxillofacial Unit, National Hospital, Abuja, Nigeria, between 2004 and 2009. There were 13 male and 10 female patients, M:F ratio 1.3:1, age range 6-62, mean (SD) 20 (13) years. The aetiological factors were trauma (n=11) that comprised falls (n=6), untreated fractures of the zygomatic arch (n=4) and myositis ossificans (n=1); infection (n=9), that comprised cancrum oris (n=3) and ear infection (n=6); congenital or unknown (n=2), and coronoid hyperplasia (n=1). The maximum interincisal distance at presentation ranged from 0 to 25 mm (mean (SD) 6.7 (7.2) mm). Seventeen had facial deformities. The diagnoses recorded were as follows: left extracapsular ankylosis, (n=8); right intracapsular bony ankylosis, (n=6); left intracapsular bony ankylosis, (n=4); bilateral intracapsular bony ankylosis, (n=4), and bilateral intracapsular fibrous ankylosis (n=1). Extreme poverty was the main predisposing factor. There is a need for a concerted effort among healthcare providers, policy makers, and the world in general to eradicate poverty and improve healthcare to limit the incidence of ankylosis of the TMJ.

  12. Airborne pollen and fungal spores in Garki, Abuja (North-Central Nigeria).

    Science.gov (United States)

    Ezike, Dimphna Nneka; Nnamani, Catherine V; Ogundipe, Oluwatoyin T; Adekanmbi, Olushola H

    2016-01-01

    The ambient atmosphere is dominated with pollen and spores, which trigger allergic reactions and diseases and impact negatively on human health. A survey of pollen and fungal spores constituents of the atmosphere of Garki, Abuja (North-Central Nigeria) was carried out for 1 year (June 1, 2011-May 31, 2012). The aim of the study was to determine the prevalence and abundance of pollen and fungal spores in the atmosphere and their relationship with meteorological parameters. Airborne samples were trapped using modified Tauber-like pollen trap, and the recipient solutions were subjected to acetolysis. Results revealed the abundance of fungal spores, pollen, fern spores, algal cysts and diatoms in decreasing order of dominance. The atmosphere was qualitatively and quantitatively dominated by pollen during the period of late rainy/harmattan season than the rainy season. Numerous fungal spores were trapped throughout the sampling periods among which Alternaria spp., Fusarium spp., Cladosporium spp. and Curvularia spp. dominated. These fungi have been implicated in allergic diseases and are dermatophytic, causing diverse skin diseases. Other pathogenic fungi found in the studied aeroflora were Dreschlera spp., Helminthosporium spp., Torula spp., Pithomyces spp., Tetraploa spp., Nigrospora ssp., Spadicoides spp., Puccinia spp. and Erysiphe graminis. Total pollen and fungal spores counts do not show significant correlation with meteorological parameters.

  13. Comparison of public and private school teachers and school principals’ opinions in Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Deniz Ozcan

    2015-01-01

    Full Text Available This study aims to examine public and private school teachers’ opinions on different aspects of their professional associations and provisions and also asks principals about teachers’ conduct and their views on the Nigerian curriculum. To conduct this study, qualitative and quantitative research models were used to investigate differences between the two organizations. Quantitative data was collected by distributing questionnaires to 118 teachers in the Abuja Municipal Area Council (AMAC, Nigeria. To conduct the qualitative analysis, 15 teachers from two schools were selected (a total of 30 teachers to answer 5 discussion questions concerning their views on the Nigerian educational system. The findings revealed that private school teachers are at an advantage when it comes to provision of resources and technology, professional development and to some degree salaries. Both public and private school teachers felt being a teacher did not bring them respect in their community. Government policy makers need to study private schools and how they operate to see how they can make changes to produce the revolutionary reform needed in education. 30 principals’ interviews revealed that public school teachers are not easily held accountable for misconduct due to the structure of leadership, while private school teachers are held accountable and any form of unprofessionalism easily leads to termination of employment.

  14. Assessment of antimicrobial drug residues in beef in Abuja, the Federal Capital Territory, Nigeria

    Directory of Open Access Journals (Sweden)

    Okwoche J. Ode

    2012-09-01

    Full Text Available Drugs administered to food-producing animals close to the time of slaughter often result in prohibited antimicrobial residues in the animal tissues at slaughter. Evidence based on the Premi® test confirmed the occurrence of antimicrobial drug residues in 89.3% of kidney and urine samples from cattle slaughtered within Abuja town where the residents rely heavily on beef as a source of protein. The administration of antibiotics close to the time of slaughter by marketers/herd owners and transporters was found to be significantly (p<0.05 higher when compared with butchers and abattoir workers. The practice of administering antibiotics to animals close to the time of slaughter was believed to be profit-motivated. The research suggests that awareness campaigns amongst the stakeholders, the enactment of appropriate laws for the control of antibiotic use and the empowerment of veterinary public health practitioners in food regulatory agencies as some of the strategies which may positively reduce the risk of antimicrobial drug residues in food animals in Nigeria.

  15. Assessment of antimicrobial drug residues in beef in Abuja, the Federal Capital Territory, Nigeria.

    Science.gov (United States)

    Omeiza, Gabriel K; Ajayi, Itopa E; Ode, Okwoche J

    2012-01-01

    Drugs administered to food-producing animals close to the time of slaughter often result in prohibited antimicrobial residues in the animal tissues at slaughter. Evidence based on the Premi® test confirmed the occurrence of antimicrobial drug residues in 89.3% of kidney and urine samples from cattle slaughtered within Abuja town where the residents rely heavily on beef as a source of protein. The administration of antibiotics close to the time of slaughter by marketers/herd owners and transporters was found to be significantly (p<0.05) higher when compared with butchers and abattoir workers. The practice of administering antibiotics to animals close to the time of slaughter was believed to be profit-motivated. The research suggests that awareness campaigns amongst the stakeholders, the enactment of appropriate laws for the control of antibiotic use and the empowerment of veterinary public health practitioners in food regulatory agencies as some of the strategies which may positively reduce the risk of antimicrobial drug residues in food animals in Nigeria.

  16. Being targeted: Young women's experience of being identified for a teenage pregnancy prevention programme.

    Science.gov (United States)

    Sorhaindo, Annik; Bonell, Chris; Fletcher, Adam; Jessiman, Patricia; Keogh, Peter; Mitchell, Kirstin

    2016-06-01

    Research on the unintended consequences of targeting 'high-risk' young people for health interventions is limited. Using qualitative data from an evaluation of the Teens & Toddlers Pregnancy Prevention programme, we explored how young women experienced being identified as at risk for teenage pregnancy to understand the processes via which unintended consequences may occur. Schools' lack of transparency regarding the targeting strategy and criteria led to feelings of confusion and mistrust among some young women. Black and minority ethnic young women perceived that the assessment of their risk was based on stereotyping. Others felt their outgoing character was misinterpreted as signifying risk. To manage these imposed labels, stigma and reputational risks, young women responded to being targeted by adopting strategies, such as distancing, silence and refusal. To limit harmful consequences, programmes could involve prospective participants in determining their need for intervention or introduce programmes for young people at all levels of risk. Copyright © 2016 The Foundation for Professionals in Services for Adolescents. Published by Elsevier Ltd. All rights reserved.

  17. Using mechanistic Bayesian networks to identify downstream targets of the sonic hedgehog pathway.

    Science.gov (United States)

    Shah, Abhik; Tenzen, Toyoaki; McMahon, Andrew P; Woolf, Peter J

    2009-12-18

    The topology of a biological pathway provides clues as to how a pathway operates, but rationally using this topology information with observed gene expression data remains a challenge. We introduce a new general-purpose analytic method called Mechanistic Bayesian Networks (MBNs) that allows for the integration of gene expression data and known constraints within a signal or regulatory pathway to predict new downstream pathway targets. The MBN framework is implemented in an open-source Bayesian network learning package, the Python Environment for Bayesian Learning (PEBL). We demonstrate how MBNs can be used by modeling the early steps of the sonic hedgehog pathway using gene expression data from different developmental stages and genetic backgrounds in mouse. Using the MBN approach we are able to automatically identify many of the known downstream targets of the hedgehog pathway such as Gas1 and Gli1, along with a short list of likely targets such as Mig12. The MBN approach shown here can easily be extended to other pathways and data types to yield a more mechanistic framework for learning genetic regulatory models.

  18. Using mechanistic Bayesian networks to identify downstream targets of the Sonic Hedgehog pathway

    Directory of Open Access Journals (Sweden)

    McMahon Andrew P

    2009-12-01

    Full Text Available Abstract Background The topology of a biological pathway provides clues as to how a pathway operates, but rationally using this topology information with observed gene expression data remains a challenge. Results We introduce a new general-purpose analytic method called Mechanistic Bayesian Networks (MBNs that allows for the integration of gene expression data and known constraints within a signal or regulatory pathway to predict new downstream pathway targets. The MBN framework is implemented in an open-source Bayesian network learning package, the Python Environment for Bayesian Learning (PEBL. We demonstrate how MBNs can be used by modeling the early steps of the sonic hedgehog pathway using gene expression data from different developmental stages and genetic backgrounds in mouse. Using the MBN approach we are able to automatically identify many of the known downstream targets of the hedgehog pathway such as Gas1 and Gli1, along with a short list of likely targets such as Mig12. Conclusions The MBN approach shown here can easily be extended to other pathways and data types to yield a more mechanistic framework for learning genetic regulatory models.

  19. Combinatorial Screening Identifies Novel Promiscuous Matrix Metalloproteinase Activities that Lead to Inhibition of the Therapeutic Target IL-13

    NARCIS (Netherlands)

    Urbach, Carole; Gordon, Nathaniel C; Strickland, Ian; Lowne, David; Joberty-Candotti, Cathy; May, Richard; Herath, Athula; Hijnen, DirkJan; Thijs, Judith L; Bruijnzeel-Koomen, Carla A; Minter, Ralph R; Hollfelder, Florian; Jermutus, Lutz

    2015-01-01

    The practical realization of disease modulation by catalytic degradation of a therapeutic target protein suffers from the difficulty to identify candidate proteases, or to engineer their specificity. We identified 23 measurable, specific, and new protease activities using combinatorial screening of

  20. Using Market Research to Characterize College Students and Identify Potential Targets for Influencing Health Behaviors.

    Science.gov (United States)

    Berg, Carla J; Ling, Pamela M; Guo, Hongfei; Windle, Michael; Thomas, Janet L; Ahluwalia, Jasjit S; An, Lawrence C

    2010-12-01

    Marketing campaigns, such as those developed by the tobacco industry, are based on market research, which defines segments of a population by assessing psychographic characteristics (i.e., attitudes, interests). This study uses a similar approach to define market segments of college smokers, to examine differences in their health behaviors (smoking, drinking, binge drinking, exercise, diet), and to determine the validity of these segments. A total of 2,265 undergraduate students aged 18-25 years completed a 108-item online survey in fall 2008 assessing demographic, psychographic (i.e., attitudes, interests), and health-related variables. Among the 753 students reporting past 30-day smoking, cluster analysis was conducted using 21 psychographic questions and identified three market segments - Stoic Individualists, Responsible Traditionalists, and Thrill-Seeking Socializers. We found that segment membership was related to frequency of alcohol use, binge drinking, and limiting dietary fat. We then developed three messages targeting each segment and conducted message testing to validate the segments on a subset of 73 smokers representing each segment in spring 2009. As hypothesized, each segment indicated greater relevance and salience for their respective message. These findings indicate that identifying qualitatively different subgroups of young adults through market research may inform the development of engaging interventions and health campaigns targeting college students.

  1. Autoantibodies targeting glomerular annexin A2 identify patients with proliferative lupus nephritis

    Science.gov (United States)

    Caster, Dawn J.; Korte, Erik A.; Merchant, Michael L.; Klein, Jon B.; Wilkey, Daniel W.; Rovin, Brad H.; Birmingham, Dan J.; Harley, John B.; Cobb, Beth L.; Namjou, Bahram; McLeish, Kenneth R.; Powell, David W.

    2015-01-01

    PURPOSE Patients with systemic lupus erythematosus (SLE) frequently develop lupus nephritis (LN), a complication frequently leading to end stage kidney disease. Immune complex deposition in the glomerulus is central to the development of LN. Using a targeted proteomic approach, we tested the hypothesis that autoantibodies targeting glomerular antigens contribute to the development of LN. EXPERIMENTAL DESIGN Human podocyte and glomerular proteins were separated by SDS-PAGE and immunoblotted with sera from SLE patients with and without LN. The regions of those gels corresponding to reactive bands observed with sera from LN patients were analyzed using LC-MS/MS. RESULTS LN reactive bands were seen at approximately 50 kDa in podocyte extracts and between 36-50 kDa in glomerular extracts. Those bands were analyzed by LC-MS/MS and 102 overlapping proteins were identified. Bioinformatic analysis determined that 36 of those proteins were membrane associated, including a protein previously suggested to contribute to glomerulonephritis and LN, annexin A2. By ELISA, patients with proliferative LN demonstrated significantly increased antibodies against annexin A2. CONCLUSION AND CLINICAL RELEVANCE Proteomic approaches identified multiple candidate antigens for autoantibodies in patients with LN. Serum antibodies against annexin A2 were significantly elevated in subjects with proliferative LN, validating those antibodies as potential biomarkers. PMID:25824007

  2. Integrative screening approach identifies regulators of polyploidization and targets for acute megakaryocytic leukemia

    Science.gov (United States)

    Wen, Qiang; Goldenson, Benjamin; Silver, Serena J.; Schenone, Monica; Dancik, Vladimir; Huang, Zan; Wang, Ling-Zhi; Lewis, Timothy; An, W. Frank; Li, Xiaoyu; Bray, Mark-Anthony; Thiollier, Clarisse; Diebold, Lauren; Gilles, Laure; Vokes, Martha S.; Moore, Christopher B.; Bliss-Moreau, Meghan; VerPlank, Lynn; Tolliday, Nicola J.; Mishra, Rama; Vemula, Sasidhar; Shi, Jianjian; Wei, Lei; Kapur, Reuben; Lopez, Cécile K.; Gerby, Bastien; Ballerini, Paola; Pflumio, Francoise; Gilliland, D. Gary; Goldberg, Liat; Birger, Yehudit; Izraeli, Shai; Gamis, Alan S.; Smith, Franklin O.; Woods, William G.; Taub, Jeffrey; Scherer, Christina A.; Bradner, James; Goh, Boon-Cher; Mercher, Thomas; Carpenter, Anne E.; Gould, Robert J.; Clemons, Paul A.; Carr, Steven A.; Root, David E.; Schreiber, Stuart L.; Stern, Andrew M.; Crispino, John D.

    2012-01-01

    Summary The mechanism by which cells decide to skip mitosis to become polyploid is largely undefined. Here we used a high-content image-based screen to identify small-molecule probes that induce polyploidization of megakaryocytic leukemia cells and serve as perturbagens to help understand this process. We found that dimethylfasudil (diMF, H-1152P) selectively increased polyploidization, mature cell-surface marker expression, and apoptosis of malignant megakaryocytes. A broadly applicable, highly integrated target identification approach employing proteomic and shRNA screening revealed that a major target of diMF is Aurora A kinase (AURKA), which has not been studied extensively in megakaryocytes. Moreover, we discovered that MLN8237 (Alisertib), a selective inhibitor of AURKA, induced polyploidization and expression of mature megakaryocyte markers in AMKL blasts and displayed potent anti-AMKL activity in vivo. This research provides the rationale to support clinical trials of MLN8237 and other inducers of polyploidization in AMKL. Finally, we have identified five networks of kinases that regulate the switch to polyploidy. PMID:22863010

  3. Using Market Research to Characterize College Students and Identify Potential Targets for Influencing Health Behaviors

    Science.gov (United States)

    Berg, Carla J.; Ling, Pamela M.; Guo, Hongfei; Windle, Michael; Thomas, Janet L.; Ahluwalia, Jasjit S.; An, Lawrence C.

    2013-01-01

    Marketing campaigns, such as those developed by the tobacco industry, are based on market research, which defines segments of a population by assessing psychographic characteristics (i.e., attitudes, interests). This study uses a similar approach to define market segments of college smokers, to examine differences in their health behaviors (smoking, drinking, binge drinking, exercise, diet), and to determine the validity of these segments. A total of 2,265 undergraduate students aged 18–25 years completed a 108-item online survey in fall 2008 assessing demographic, psychographic (i.e., attitudes, interests), and health-related variables. Among the 753 students reporting past 30-day smoking, cluster analysis was conducted using 21 psychographic questions and identified three market segments – Stoic Individualists, Responsible Traditionalists, and Thrill-Seeking Socializers. We found that segment membership was related to frequency of alcohol use, binge drinking, and limiting dietary fat. We then developed three messages targeting each segment and conducted message testing to validate the segments on a subset of 73 smokers representing each segment in spring 2009. As hypothesized, each segment indicated greater relevance and salience for their respective message. These findings indicate that identifying qualitatively different subgroups of young adults through market research may inform the development of engaging interventions and health campaigns targeting college students. PMID:25264429

  4. Identifying genetic modulators of the connectivity between transcription factors and their transcriptional targets.

    Science.gov (United States)

    Fazlollahi, Mina; Muroff, Ivor; Lee, Eunjee; Causton, Helen C; Bussemaker, Harmen J

    2016-03-29

    Regulation of gene expression by transcription factors (TFs) is highly dependent on genetic background and interactions with cofactors. Identifying specific context factors is a major challenge that requires new approaches. Here we show that exploiting natural variation is a potent strategy for probing functional interactions within gene regulatory networks. We developed an algorithm to identify genetic polymorphisms that modulate the regulatory connectivity between specific transcription factors and their target genes in vivo. As a proof of principle, we mapped connectivity quantitative trait loci (cQTLs) using parallel genotype and gene expression data for segregants from a cross between two strains of the yeast Saccharomyces cerevisiae We identified a nonsynonymous mutation in the DIG2 gene as a cQTL for the transcription factor Ste12p and confirmed this prediction empirically. We also identified three polymorphisms in TAF13 as putative modulators of regulation by Gcn4p. Our method has potential for revealing how genetic differences among individuals influence gene regulatory networks in any organism for which gene expression and genotype data are available along with information on binding preferences for transcription factors.

  5. The potential of metatranscriptomics for identifying screening targets for bacterial vaginosis.

    Directory of Open Access Journals (Sweden)

    Jimmy Twin

    Full Text Available BACKGROUND: The ribosomal RNA content of a sample collected from a woman with bacterial vaginosis (BV was analysed to determine the active microbial community, and to identify potential targets for further screening. METHODOLOGY/PRINCIPAL FINDINGS: The sample from the BV patient underwent total RNA extraction, followed by physical subtraction of human rRNA and whole transcriptome amplification. The metatranscriptome was sequenced using Roche 454 titanium chemistry. The bioinformatics pipeline MG-RAST and desktop DNA analysis platforms were utilised to analyse results. Bacteria of the genus Prevotella (predominately P. amnii constituted 36% of the 16S rRNA reads, followed by Megasphaera (19%, Leptotrichia/Sneathia (8% and Fusobacterium (8%. Comparison of the abundances of several bacteria to quantitative PCR (qPCR screening of extracted DNA revealed comparable relative abundances. This suggests a correlation between what was present and transcriptionally active in this sample: however distinct differences were seen when compared to the microbiome determined by 16S rRNA gene amplicon sequencing. To assess the presence of P. amnii in a larger pool of samples, 90 sexually active women were screened using qPCR. This bacterium was found to be strongly associated with BV (P<0.001, OR 23.3 (95%CI:2.9-190.7 among the 90 women. CONCLUSIONS/SIGNIFICANCE: This study highlighted the potential of metatranscriptomics as a tool for characterising metabolically active microbiota and identifying targets for further screening. Prevotella amnii was chosen as an example target, being the most metabolically active species present in the single patient with BV, and was found to be detected at a high concentration by qPCR in 31% of cohort with BV, with an association with both oral and penile-vaginal sex.

  6. Identifying co-targets to fight drug resistance based on a random walk model

    Directory of Open Access Journals (Sweden)

    Chen Liang-Chun

    2012-01-01

    Full Text Available Abstract Background Drug resistance has now posed more severe and emergent threats to human health and infectious disease treatment. However, wet-lab approaches alone to counter drug resistance have so far still achieved limited success due to less knowledge about the underlying mechanisms of drug resistance. Our approach apply a heuristic search algorithm in order to extract active network under drug treatment and use a random walk model to identify potential co-targets for effective antibacterial drugs. Results We use interactome network of Mycobacterium tuberculosis and gene expression data which are treated with two kinds of antibiotic, Isoniazid and Ethionamide as our test data. Our analysis shows that the active drug-treated networks are associated with the trigger of fatty acid metabolism and synthesis and nicotinamide adenine dinucleotide (NADH-related processes and those results are consistent with the recent experimental findings. Efflux pumps processes appear to be the major mechanisms of resistance but SOS response is significantly up-regulation under Isoniazid treatment. We also successfully identify the potential co-targets with literature confirmed evidences which are related to the glycine-rich membrane, adenosine triphosphate energy and cell wall processes. Conclusions With gene expression and interactome data supported, our study points out possible pathways leading to the emergence of drug resistance under drug treatment. We develop a computational workflow for giving new insights to bacterial drug resistance which can be gained by a systematic and global analysis of the bacterial regulation network. Our study also discovers the potential co-targets with good properties in biological and graph theory aspects to overcome the problem of drug resistance.

  7. Quantitative liver proteomics identifies FGF19 targets that couple metabolism and proliferation

    Science.gov (United States)

    Vos, Harmjan R.; Burgering, Boudewijn M. T.; van Mil, Saskia W. C.

    2017-01-01

    Fibroblast growth factor 19 (FGF19) is a gut-derived peptide hormone that is produced following activation of Farnesoid X Receptor (FXR). FGF19 is secreted and signals to the liver, where it contributes to the homeostasis of bile acid (BA), lipid and carbohydrate metabolism. FGF19 is a promising therapeutic target for the metabolic syndrome and cholestatic diseases, but enthusiasm for its use has been tempered by FGF19-mediated induction of proliferation and hepatocellular carcinoma. To inform future rational design of FGF19-variants, we have conducted temporal quantitative proteomic and gene expression analyses to identify FGF19-targets related to metabolism and proliferation. Mice were fasted for 16 hours, and injected with human FGF19 (1 mg/kg body weight) or vehicle. Liver protein extracts (containing “light” lysine) were mixed 1:1 with a spike-in protein extract from 13C6-lysine metabolically labelled mouse liver (containing “heavy” lysine) and analysed by LC-MS/MS. Our analyses provide a resource of FGF19 target proteins in the liver. 189 proteins were upregulated (≥ 1.5 folds) and 73 proteins were downregulated (≤ -1.5 folds) by FGF19. FGF19 treatment decreased the expression of proteins involved in fatty acid (FA) synthesis, i.e., Fabp5, Scd1, and Acsl3 and increased the expression of Acox1, involved in FA oxidation. As expected, FGF19 increased the expression of proteins known to drive proliferation (i.e., Tgfbi, Vcam1, Anxa2 and Hdlbp). Importantly, many of the FGF19 targets (i.e., Pdk4, Apoa4, Fas and Stat3) have a dual function in both metabolism and cell proliferation. Therefore, our findings challenge the development of FGF19-variants that fully uncouple metabolic benefit from mitogenic potential. PMID:28178326

  8. Quantitative liver proteomics identifies FGF19 targets that couple metabolism and proliferation.

    Science.gov (United States)

    Massafra, Vittoria; Milona, Alexandra; Vos, Harmjan R; Burgering, Boudewijn M T; van Mil, Saskia W C

    2017-01-01

    Fibroblast growth factor 19 (FGF19) is a gut-derived peptide hormone that is produced following activation of Farnesoid X Receptor (FXR). FGF19 is secreted and signals to the liver, where it contributes to the homeostasis of bile acid (BA), lipid and carbohydrate metabolism. FGF19 is a promising therapeutic target for the metabolic syndrome and cholestatic diseases, but enthusiasm for its use has been tempered by FGF19-mediated induction of proliferation and hepatocellular carcinoma. To inform future rational design of FGF19-variants, we have conducted temporal quantitative proteomic and gene expression analyses to identify FGF19-targets related to metabolism and proliferation. Mice were fasted for 16 hours, and injected with human FGF19 (1 mg/kg body weight) or vehicle. Liver protein extracts (containing "light" lysine) were mixed 1:1 with a spike-in protein extract from 13C6-lysine metabolically labelled mouse liver (containing "heavy" lysine) and analysed by LC-MS/MS. Our analyses provide a resource of FGF19 target proteins in the liver. 189 proteins were upregulated (≥ 1.5 folds) and 73 proteins were downregulated (≤ -1.5 folds) by FGF19. FGF19 treatment decreased the expression of proteins involved in fatty acid (FA) synthesis, i.e., Fabp5, Scd1, and Acsl3 and increased the expression of Acox1, involved in FA oxidation. As expected, FGF19 increased the expression of proteins known to drive proliferation (i.e., Tgfbi, Vcam1, Anxa2 and Hdlbp). Importantly, many of the FGF19 targets (i.e., Pdk4, Apoa4, Fas and Stat3) have a dual function in both metabolism and cell proliferation. Therefore, our findings challenge the development of FGF19-variants that fully uncouple metabolic benefit from mitogenic potential.

  9. Targeting the IL-6 dependent phenotype can identify novel therapies for cholangiocarcinoma.

    Directory of Open Access Journals (Sweden)

    Chiara Braconi

    Full Text Available BACKGROUND: The need for new therapies for cholangiocarcinoma is highlighted by their poor prognosis and refractoriness to chemotherapy. Increased production of Interleukin-6 promotes cholangiocarcinoma growth and contributes to chemoresistance by activating cell survival mechanisms. We sought to identify biologically active compounds capable of ameliorating the phenotypic effects of IL-6 expression and to explore their potential therapeutic use for cholangiocarcinoma. METHODOLOGY: A genomic signature associated with Interleukin-6 expression in Mz-ChA-1 human malignant cholangiocytes was derived. Computational bioinformatics analysis was performed to identify compounds that induced inverse gene changes to the signature. The effect of these compounds on cholangiocarcinoma growth was then experimentally verified in vitro and in vivo. Interactions with other therapeutic agents were evaluated using median effects analysis. PRINCIPAL FINDINGS: A group of structurally related compounds, nitrendipine, nifedipine and felodipine was identified. All three compounds were cytotoxic to Mz-ChA-1 cells with an IC50 for felodipine of 26 µM, nitrendipine, 44 µM and nifedipine, 15 µM. Similar results were observed in KMCH-1, CC-LP-1 and TFK-1 cholangiocarcinoma cell lines. At a fractional effect of 0.5, all three agents were synergistic with either camptothecin or gemcitabine in Mz-ChA-1 cells in vitro. Co-administration of felodipine and gemcitabine decreased the growth of Mz-ChA-1 cell xenografts in nude athymic mice. CONCLUSIONS: Computational bioinformatics analysis of phenotype-based genomic expression can be used to identify therapeutic agents. Using this drug discovery approach based on targeting a defined tumor associated phenotype, we identified compounds with the potential for therapeutic use in cholangiocarcinoma.

  10. Targeted next generation sequencing of mucosal melanomas identifies frequent NF1 and RAS mutations.

    Science.gov (United States)

    Cosgarea, Ioana; Ugurel, Selma; Sucker, Antje; Livingstone, Elisabeth; Zimmer, Lisa; Ziemer, Mirjana; Utikal, Jochen; Mohr, Peter; Pfeiffer, Christiane; Pföhler, Claudia; Hillen, Uwe; Horn, Susanne; Schadendorf, Dirk; Griewank, Klaus G; Roesch, Alexander

    2017-06-20

    Mucosal melanoma represents ~1% of all melanomas, frequently having a poor prognosis due to diagnosis at a late stage of disease. Mucosal melanoma differs from cutaneous melanoma not only in terms of poorer clinical outcome but also on the molecular level having e.g. less BRAF and more frequent KIT mutations than cutaneous melanomas. For the majority of mucosal melanomas oncogenic driver mutations remain unknown. In our study, 75 tumor tissues from patients diagnosed with mucosal melanoma were analyzed, applying a targeted next generation sequencing panel covering 29 known recurrently mutated genes in melanoma. NF1 and RAS mutations were identified as the most frequently mutated genes occurring in 18.3% and 16.9% of samples, respectively. Mutations in BRAF were identified in 8.4% and KIT in 7.0% of tumor samples. Our study identifies NF1 as the most frequently occurring driver mutation in mucosal melanoma. RAS alterations, consisting of NRAS and KRAS mutations, were the second most frequent mutation type. BRAF and KIT mutations were rare with frequencies below 10% each. Our data indicate that in mucosal melanomas RAS/NF1 alterations are frequent, implying a significant pathogenetic role for MAPK and potentially PI3K pathway activation in these tumors.

  11. Identifying Human Genome-Wide CNV, LOH and UPD by Targeted Sequencing of Selected Regions.

    Directory of Open Access Journals (Sweden)

    Wei Li

    Full Text Available Copy-number variations (CNV, loss of heterozygosity (LOH, and uniparental disomy (UPD are large genomic aberrations leading to many common inherited diseases, cancers, and other complex diseases. An integrated tool to identify these aberrations is essential in understanding diseases and in designing clinical interventions. Previous discovery methods based on whole-genome sequencing (WGS require very high depth of coverage on the whole genome scale, and are cost-wise inefficient. Another approach, whole exome genome sequencing (WEGS, is limited to discovering variations within exons. Thus, we are lacking efficient methods to detect genomic aberrations on the whole genome scale using next-generation sequencing technology. Here we present a method to identify genome-wide CNV, LOH and UPD for the human genome via selectively sequencing a small portion of genome termed Selected Target Regions (SeTRs. In our experiments, the SeTRs are covered by 99.73%~99.95% with sufficient depth. Our developed bioinformatics pipeline calls genome-wide CNVs with high confidence, revealing 8 credible events of LOH and 3 UPD events larger than 5M from 15 individual samples. We demonstrate that genome-wide CNV, LOH and UPD can be detected using a cost-effective SeTRs sequencing approach, and that LOH and UPD can be identified using just a sample grouping technique, without using a matched sample or familial information.

  12. Quantitative proteomics identify molecular targets that are crucial in larval settlement and metamorphosis of bugula neritina

    KAUST Repository

    Zhang, Huoming

    2011-01-07

    The marine invertebrate Bugula neritina has a biphasic life cycle that consists of a swimming larval stage and a sessile juvenile and adult stage. The attachment of larvae to the substratum and their subsequent metamorphosis have crucial ecological consequences. Despite many studies on this species, little is known about the molecular mechanism of these processes. Here, we report a comparative study of swimming larvae and metamorphosing individuals at 4 and 24 h postattachment using label-free quantitative proteomics. We identified more than 1100 proteins at each stage, 61 of which were differentially expressed. Specifically, proteins involved in energy metabolism and structural molecules were generally down-regulated, whereas proteins involved in transcription and translation, the extracellular matrix, and calcification were strongly up-regulated during metamorphosis. Many tightly regulated novel proteins were also identified. Subsequent analysis of the temporal and spatial expressions of some of the proteins and an assay of their functions indicated that they may have key roles in metamorphosis of B. neritina. These findings not only provide molecular evidence with which to elucidate the substantial changes in morphology and physiology that occur during larval attachment and metamorphosis but also identify potential targets for antifouling treatment. © 2011 American Chemical Society.

  13. Identifying Human Genome-Wide CNV, LOH and UPD by Targeted Sequencing of Selected Regions.

    Science.gov (United States)

    Wang, Yu; Li, Wei; Xia, Yingying; Wang, Chongzhi; Tang, Y Tom; Guo, Wenying; Li, Jinliang; Zhao, Xia; Sun, Yepeng; Hu, Juan; Zhen, Hefu; Zhang, Xiandong; Chen, Chao; Shi, Yujian; Li, Lin; Cao, Hongzhi; Du, Hongli; Li, Jian

    2014-01-01

    Copy-number variations (CNV), loss of heterozygosity (LOH), and uniparental disomy (UPD) are large genomic aberrations leading to many common inherited diseases, cancers, and other complex diseases. An integrated tool to identify these aberrations is essential in understanding diseases and in designing clinical interventions. Previous discovery methods based on whole-genome sequencing (WGS) require very high depth of coverage on the whole genome scale, and are cost-wise inefficient. Another approach, whole exome genome sequencing (WEGS), is limited to discovering variations within exons. Thus, we are lacking efficient methods to detect genomic aberrations on the whole genome scale using next-generation sequencing technology. Here we present a method to identify genome-wide CNV, LOH and UPD for the human genome via selectively sequencing a small portion of genome termed Selected Target Regions (SeTRs). In our experiments, the SeTRs are covered by 99.73%~99.95% with sufficient depth. Our developed bioinformatics pipeline calls genome-wide CNVs with high confidence, revealing 8 credible events of LOH and 3 UPD events larger than 5M from 15 individual samples. We demonstrate that genome-wide CNV, LOH and UPD can be detected using a cost-effective SeTRs sequencing approach, and that LOH and UPD can be identified using just a sample grouping technique, without using a matched sample or familial information.

  14. Genetic Variants Identified from Epilepsy of Unknown Etiology in Chinese Children by Targeted Exome Sequencing

    Science.gov (United States)

    Wang, Yimin; Du, Xiaonan; Bin, Rao; Yu, Shanshan; Xia, Zhezhi; Zheng, Guo; Zhong, Jianmin; Zhang, Yunjian; Jiang, Yong-hui; Wang, Yi

    2017-01-01

    Genetic factors play a major role in the etiology of epilepsy disorders. Recent genomics studies using next generation sequencing (NGS) technique have identified a large number of genetic variants including copy number (CNV) and single nucleotide variant (SNV) in a small set of genes from individuals with epilepsy. These discoveries have contributed significantly to evaluate the etiology of epilepsy in clinic and lay the foundation to develop molecular specific treatment. However, the molecular basis for a majority of epilepsy patients remains elusive, and furthermore, most of these studies have been conducted in Caucasian children. Here we conducted a targeted exome-sequencing of 63 trios of Chinese epilepsy families using a custom-designed NGS panel that covers 412 known and candidate genes for epilepsy. We identified pathogenic and likely pathogenic variants in 15 of 63 (23.8%) families in known epilepsy genes including SCN1A, CDKL5, STXBP1, CHD2, SCN3A, SCN9A, TSC2, MBD5, POLG and EFHC1. More importantly, we identified likely pathologic variants in several novel candidate genes such as GABRE, MYH1, and CLCN6. Our results provide the evidence supporting the application of custom-designed NGS panel in clinic and indicate a conserved genetic susceptibility for epilepsy between Chinese and Caucasian children. PMID:28074849

  15. Female resistance to pneumonia identifies lung macrophage nitric oxide synthase-3 as a therapeutic target

    Science.gov (United States)

    Yang, Zhiping; Huang, Yuh-Chin T; Koziel, Henry; de Crom, Rini; Ruetten, Hartmut; Wohlfart, Paulus; Thomsen, Reimar W; Kahlert, Johnny A; Sørensen, Henrik Toft; Jozefowski, Szczepan; Colby, Amy; Kobzik, Lester

    2014-01-01

    To identify new approaches to enhance innate immunity to bacterial pneumonia, we investigated the natural experiment of gender differences in resistance to infections. Female and estrogen-treated male mice show greater resistance to pneumococcal pneumonia, seen as greater bacterial clearance, diminished lung inflammation, and better survival. In vitro, lung macrophages from female mice and humans show better killing of ingested bacteria. Inhibitors and genetically altered mice identify a critical role for estrogen-mediated activation of lung macrophage nitric oxide synthase-3 (NOS3). Epidemiologic data show decreased hospitalization for pneumonia in women receiving estrogen or statins (known to activate NOS3). Pharmacologic targeting of NOS3 with statins or another small-molecule compound (AVE3085) enhanced macrophage bacterial killing, improved bacterial clearance, and increased host survival in both primary and secondary (post-influenza) pneumonia. The data identify a novel mechanism for host defense via NOS3 and suggest a potential therapeutic strategy to reduce secondary bacterial pneumonia after influenza. DOI: http://dx.doi.org/10.7554/eLife.03711.001 PMID:25317947

  16. Targeting the Akt1 allosteric site to identify novel scaffolds through virtual screening.

    Science.gov (United States)

    Yilmaz, Oya Gursoy; Olmez, Elif Ozkirimli; Ulgen, Kutlu O

    2014-02-01

    Preclinical data and tumor specimen studies report that AKT kinases are related to many human cancers. Therefore, identification and development of small molecule inhibitors targeting AKT and its signaling pathway can be therapeutic in treatment of cancer. Numerous studies report inhibitors that target the ATP-binding pocket in the kinase domains, but the similarity of this site, within the kinase family makes selectivity a major problem. The sequence identity amongst PH domains is significantly lower than that in kinase domains and developing more selective inhibitors is possible if PH domain is targeted. This in silico screening study is the first time report toward the identification of potential allosteric inhibitors expected to bind the cavity between kinase and PH domains of Akt1. Structural information of Akt1 was used to develop structure-based pharmacophore models comprising hydrophobic, acceptor, donor and ring features. The 3D structural information of previously identified allosteric Akt inhibitors obtained from literature was employed to develop a ligand-based pharmacophore model. Database was generated with drug like subset of ZINC and screening was performed based on 3D similarity to the selected pharmacophore hypotheses. Binding modes and affinities of the ligands were predicted by Glide software. Top scoring hits were further analyzed considering 2D similarity between the compounds, interactions with Akt1, fitness to pharmacophore models, ADME, druglikeness criteria and Induced-Fit docking. Using virtual screening methodologies, derivatives of 3-methyl-xanthine, quinoline-4-carboxamide and 2-[4-(cyclohexa-1,3-dien-1-yl)-1H-pyrazol-3-yl]phenol were proposed as potential leads for allosteric inhibition of Akt1.

  17. Haploinsufficiency networks identify targetable patterns of allelic deficiency in low mutation ovarian cancer

    Science.gov (United States)

    Delaney, Joe Ryan; Patel, Chandni B.; Willis, Katelyn McCabe; Haghighiabyaneh, Mina; Axelrod, Joshua; Tancioni, Isabelle; Lu, Dan; Bapat, Jaidev; Young, Shanique; Cadassou, Octavia; Bartakova, Alena; Sheth, Parthiv; Haft, Carley; Hui, Sandra; Saenz, Cheryl; Schlaepfer, David D.; Harismendy, Olivier; Stupack, Dwayne G.

    2017-01-01

    Identification of specific oncogenic gene changes has enabled the modern generation of targeted cancer therapeutics. In high-grade serous ovarian cancer (OV), the bulk of genetic changes is not somatic point mutations, but rather somatic copy-number alterations (SCNAs). The impact of SCNAs on tumour biology remains poorly understood. Here we build haploinsufficiency network analyses to identify which SCNA patterns are most disruptive in OV. Of all KEGG pathways (N=187), autophagy is the most significantly disrupted by coincident gene deletions. Compared with 20 other cancer types, OV is most severely disrupted in autophagy and in compensatory proteostasis pathways. Network analysis prioritizes MAP1LC3B (LC3) and BECN1 as most impactful. Knockdown of LC3 and BECN1 expression confers sensitivity to cells undergoing autophagic stress independent of platinum resistance status. The results support the use of pathway network tools to evaluate how the copy-number landscape of a tumour may guide therapy. PMID:28198375

  18. A genetic strategy to identify targets for the development of drugs that prevent bacterial persistence.

    Science.gov (United States)

    Kim, Jee-Hyun; O'Brien, Kathryn M; Sharma, Ritu; Boshoff, Helena I M; Rehren, German; Chakraborty, Sumit; Wallach, Joshua B; Monteleone, Mercedes; Wilson, Daniel J; Aldrich, Courtney C; Barry, Clifton E; Rhee, Kyu Y; Ehrt, Sabine; Schnappinger, Dirk

    2013-11-19

    Antibacterial drug development suffers from a paucity of targets whose inhibition kills replicating and nonreplicating bacteria. The latter include phenotypically dormant cells, known as persisters, which are tolerant to many antibiotics and often contribute to failure in the treatment of chronic infections. This is nowhere more apparent than in tuberculosis caused by Mycobacterium tuberculosis, a pathogen that tolerates many antibiotics once it ceases to replicate. We developed a strategy to identify proteins that Mycobacterium tuberculosis requires to both grow and persist and whose inhibition has the potential to prevent drug tolerance and persister formation. This strategy is based on a tunable dual-control genetic switch that provides a regulatory range spanning three orders of magnitude, quickly depletes proteins in both replicating and nonreplicating mycobacteria, and exhibits increased robustness to phenotypic reversion. Using this switch, we demonstrated that depletion of the nicotinamide adenine dinucleotide synthetase (NadE) rapidly killed Mycobacterium tuberculosis under conditions of standard growth and nonreplicative persistence induced by oxygen and nutrient limitation as well as during the acute and chronic phases of infection in mice. These findings establish the dual-control switch as a robust tool with which to probe the essentiality of Mycobacterium tuberculosis proteins under different conditions, including those that induce antibiotic tolerance, and NadE as a target with the potential to shorten current tuberculosis chemotherapies.

  19. Differences between visual hemifields in identifying rapidly presented target stimuli: Letters and digits, faces, and shapes.

    Directory of Open Access Journals (Sweden)

    Dariusz eAsanowicz

    2013-07-01

    Full Text Available The right hemisphere has been shown to play a dominant role in processing of visuo-spatial information. Recently, this role has been studied in the two-stream rapid serial visual presentation task. In this task, two alphanumerical targets are embedded in left and right simultaneous streams of rapidly changing letters. The second target (T2 is identified better in the left than in the right visual field. This difference has been interpreted as advantage of the right hemisphere (RH. However, a disadvantage of the left hemisphere (LH could not be excluded so far. The LH, specialized for processing of verbal stimuli, might be overloaded due to constant input of letters from both visual fields. In the present study, this overload hypothesis was tested by reducing demands on verbal processing (Experiments 1, and by overloading the RH with nonverbal stimuli: faces (Experiment 2 and irregular shapes (Experiment 3. The left visual field advantage proved to be largely independent from the level of verbal load and from stimulus type. Therefore, although not entirely disproving the overload hypothesis, these results suggest as the most parsimonious explanation this asymmetry reflects a RH advantage, presumably in perceptual and attentional processing, rather than a LH disadvantage caused by verbal overload.

  20. Multiplatform molecular profiling identifies potentially targetable biomarkers in malignant phyllodes tumors of the breast.

    Science.gov (United States)

    Gatalica, Zoran; Vranic, Semir; Ghazalpour, Anatole; Xiu, Joanne; Ocal, Idris Tolgay; McGill, John; Bender, Ryan P; Discianno, Erin; Schlum, Aaron; Sanati, Souzan; Palazzo, Juan; Reddy, Sandeep; Pockaj, Barbara

    2016-01-12

    Malignant phyllodes tumor is a rare breast malignancy with sarcomatous overgrowth and with limited effective treatment options for recurrent and metastatic cases. Recent clinical trials indicated a potential for anti-angiogenic, anti-EGFR and immunotherapeutic approaches for patients with sarcomas, which led us to investigate these and other targetable pathways in malignant phyllodes tumor of the breast. Thirty-six malignant phyllodes tumors (including 8 metastatic tumors with two cases having matched primary and metastatic tumors) were profiled using gene sequencing, gene copy number analysis, whole genome expression, and protein expression. Whole genome expression analysis demonstrated consistent over-expression of genes involved in angiogenesis including VEGFA, Angiopoietin-2, VCAM1, PDGFRA, and PTTG1. EGFR protein overexpression was observed in 26/27 (96%) of cases with amplification of the EGFR gene in 8/24 (33%) cases. Two EGFR mutations were identified including EGFRvIII and a presumed pathogenic V774M mutation, respectively. The most common pathogenic mutations included TP53 (50%) and PIK3CA (15%). Cases with matched primary and metastatic tumors harbored identical mutations in both sites (PIK3CA/KRAS and RB1 gene mutations, respectively). Tumor expression of PD-L1 immunoregulatory protein was observed in 3/22 (14%) of cases. Overexpression of molecular biomarkers of increased angiogenesis, EGFR and immune checkpoints provides novel targeted therapy options in malignant phyllodes tumors of the breast.

  1. Identifying On-Orbit Test Targets for Space Fence Operational Testing

    Science.gov (United States)

    Pechkis, D.; Pacheco, N.; Botting, T.

    2014-09-01

    Space Fence will be an integrated system of two ground-based, S-band (2 to 4 GHz) phased-array radars located in Kwajalein and perhaps Western Australia [1]. Space Fence will cooperate with other Space Surveillance Network sensors to provide space object tracking and radar characterization data to support U.S. Strategic Command space object catalog maintenance and other space situational awareness needs. We present a rigorous statistical test design intended to test Space Fence to the letter of the program requirements as well as to characterize the system performance across the entire operational envelope. The design uses altitude, size, and inclination as independent factors in statistical tests of dependent variables (e.g., observation accuracy) linked to requirements. The analysis derives the type and number of necessary test targets. Comparing the resulting sample sizes with the number of currently known targets, we identify those areas where modelling and simulation methods are needed. Assuming hypothetical Kwajalein radar coverage and a conservative number of radar passes per object per day, we conclude that tests involving real-world space objects should take no more than 25 days to evaluate all operational requirements; almost 60 percent of the requirements can be tested in a single day and nearly 90 percent can be tested in one week or less. Reference: [1] L. Haines and P. Phu, Space Fence PDR Concept Development Phase, 2011 AMOS Conference Technical Papers.

  2. Controllability analysis of the directed human protein interaction network identifies disease genes and drug targets.

    Science.gov (United States)

    Vinayagam, Arunachalam; Gibson, Travis E; Lee, Ho-Joon; Yilmazel, Bahar; Roesel, Charles; Hu, Yanhui; Kwon, Young; Sharma, Amitabh; Liu, Yang-Yu; Perrimon, Norbert; Barabási, Albert-László

    2016-05-03

    The protein-protein interaction (PPI) network is crucial for cellular information processing and decision-making. With suitable inputs, PPI networks drive the cells to diverse functional outcomes such as cell proliferation or cell death. Here, we characterize the structural controllability of a large directed human PPI network comprising 6,339 proteins and 34,813 interactions. This network allows us to classify proteins as "indispensable," "neutral," or "dispensable," which correlates to increasing, no effect, or decreasing the number of driver nodes in the network upon removal of that protein. We find that 21% of the proteins in the PPI network are indispensable. Interestingly, these indispensable proteins are the primary targets of disease-causing mutations, human viruses, and drugs, suggesting that altering a network's control property is critical for the transition between healthy and disease states. Furthermore, analyzing copy number alterations data from 1,547 cancer patients reveals that 56 genes that are frequently amplified or deleted in nine different cancers are indispensable. Among the 56 genes, 46 of them have not been previously associated with cancer. This suggests that controllability analysis is very useful in identifying novel disease genes and potential drug targets.

  3. Targeting Policy for Obesity Prevention: Identifying the Critical Age for Weight Gain in Women

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    Trevor J. B. Dummer

    2012-01-01

    Full Text Available The obesity epidemic requires the development of prevention policy targeting individuals most likely to benefit. We used self-reported prepregnancy body weight of all women giving birth in Nova Scotia between 1988 and 2006 to define obesity and evaluated socioeconomic, demographic, and temporal trends in obesity using linear regression. There were 172,373 deliveries in this cohort of 110,743 women. Maternal body weight increased significantly by 0.5 kg per year from 1988, and lower income and rural residence were both associated significantly with increasing obesity. We estimated an additional 82,000 overweight or obese women in Nova Scotia in 2010, compared to the number that would be expected from obesity rates of just two decades ago. The critical age for weight gain was identified as being between 20 and 24 years. This age group is an important transition age between adolescence and adulthood when individuals first begin to accept responsibility for food planning, purchasing, and preparation. Policy and public health interventions must target those most at risk, namely, younger women and the socially deprived, whilst tackling the marketing of low-cost energy-dense foods at the expense of healthier options.

  4. OptForce: an optimization procedure for identifying all genetic manipulations leading to targeted overproductions.

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    Sridhar Ranganathan

    2010-04-01

    Full Text Available Computational procedures for predicting metabolic interventions leading to the overproduction of biochemicals in microbial strains are widely in use. However, these methods rely on surrogate biological objectives (e.g., maximize growth rate or minimize metabolic adjustments and do not make use of flux measurements often available for the wild-type strain. In this work, we introduce the OptForce procedure that identifies all possible engineering interventions by classifying reactions in the metabolic model depending upon whether their flux values must increase, decrease or become equal to zero to meet a pre-specified overproduction target. We hierarchically apply this classification rule for pairs, triples, quadruples, etc. of reactions. This leads to the identification of a sufficient and non-redundant set of fluxes that must change (i.e., MUST set to meet a pre-specified overproduction target. Starting with this set we subsequently extract a minimal set of fluxes that must actively be forced through genetic manipulations (i.e., FORCE set to ensure that all fluxes in the network are consistent with the overproduction objective. We demonstrate our OptForce framework for succinate production in Escherichia coli using the most recent in silico E. coli model, iAF1260. The method not only recapitulates existing engineering strategies but also reveals non-intuitive ones that boost succinate production by performing coordinated changes on pathways distant from the last steps of succinate synthesis.

  5. HLA ligandomics identifies histone deacetylase 1 as target for ovarian cancer immunotherapy.

    Science.gov (United States)

    Peper, Janet Kerstin; Bösmüller, Hans-Christian; Schuster, Heiko; Gückel, Brigitte; Hörzer, Helen; Roehle, Kevin; Schäfer, Richard; Wagner, Philipp; Rammensee, Hans-Georg; Stevanović, Stefan; Fend, Falko; Staebler, Annette

    2016-05-01

    The recent approval of clincially effective immune checkpoint inhibitors illustrates the potential of cancer immunotherapy. A challenging task remains the identification of specific targets guiding immunotherapy. Facilitated by technical advances, the direct identification of physiologically relevant targets is enabled by analyzing the HLA ligandome of cancer cells. Since recent publications demonstrate the immunogenicity of ovarian cancer (OvCa), immunotherapies, including peptide-based cancer vaccines, represent a promising treatment approach. To identify vaccine peptides, we employed a combined strategy of HLA ligandomics in high-grade serous OvCa samples and immunogenicity analysis. Only few proteins were naturally presented as HLA ligands on all samples analyzed, including histone deacetylase (HDAC) 1 and 2. In vitro priming of CD8(+) T cells demonstrated that two HDAC1/2-derived HLA ligands can induce T-cell responses, capable of killing HLA-matched tumor cells. High HDAC1 expression shown by immunohistochemistry in 136 high-grade serous OvCa patients associated with significantly reduced overall survival (OS), whereas patients with high numbers of CD3(+) tumor-infiltrating lymphocytes (TILs) in the tumor epithelium and CD8(+) TILs in the tumor stroma showed improved OS. However, correlating HDAC1 expression with TILs, high levels of TILs abrogated the impact of HDAC1 on OS. This study strengthens the role of HDAC1/2 as an important tumor antigen in OvCa, demonstrating its impact on OS in a large cohort of OvCa patients. We further identified two immunogenic HDAC1-derived peptides, which frequently induce multi-functional T-cell responses in many donors, suitable for future multi-peptide vaccine trials in OvCa patients.

  6. Correlation of phenotypic profiles using targeted proteomics identifies mycobacterial esx-1 substrates.

    Science.gov (United States)

    Champion, Matthew M; Williams, Emily A; Pinapati, Richard S; Champion, Patricia A DiGiuseppe

    2014-11-07

    The Esx/WXG-100 (ESAT-6/Wss) exporters are multiprotein complexes that promote protein translocation across the cytoplasmic membrane in a diverse range of pathogenic and nonpathogenic bacterial species. The Esx-1 (ESAT-6 System-1) system mediates virulence factor translocation in mycobacterial pathogens, including the human pathogen Mycobacterium tuberculosis. Although several genes have been associated with Esx-1-mediated transport and virulence, the contribution of individual Esx-1 genes to export is largely undefined. A unique aspect of Esx-1 export is that several substrates require each other for export/stability. We exploited substrate "codependency" to identify Esx-1 substrates. We simultaneously quantified changes in the levels of 13 Esx-1 proteins from both secreted and cytosolic protein fractions generated from 16 Esx-1-deficient Mycobacterium marinum strains in a single experiment using MRM/SRM targeted mass spectrometry. This expansion of measurable Esx-1 proteins allowed us to define statistical rules for assigning novel substrates using phenotypic profiles of known Esx-1 substrates. Using this approach, we identified three additional Esx-1 substrates encoded by the esx-1 region. Our studies begin to address how disruption of specific genes affects several proteins in the Esx-1 complex. Overall, our findings illuminate relationships between Esx-1 proteins and create a framework for the identification of secreted substrates applicable to other protein exporters and pathways.

  7. Correlation of Phenotypic Profiles Using Targeted Proteomics Identifies Mycobacterial Esx-1 Substrates

    Science.gov (United States)

    2015-01-01

    The Esx/WXG-100 (ESAT-6/Wss) exporters are multiprotein complexes that promote protein translocation across the cytoplasmic membrane in a diverse range of pathogenic and nonpathogenic bacterial species. The Esx-1 (ESAT-6 System-1) system mediates virulence factor translocation in mycobacterial pathogens, including the human pathogen Mycobacterium tuberculosis. Although several genes have been associated with Esx-1-mediated transport and virulence, the contribution of individual Esx-1 genes to export is largely undefined. A unique aspect of Esx-1 export is that several substrates require each other for export/stability. We exploited substrate “codependency” to identify Esx-1 substrates. We simultaneously quantified changes in the levels of 13 Esx-1 proteins from both secreted and cytosolic protein fractions generated from 16 Esx-1-deficient Mycobacterium marinum strains in a single experiment using MRM/SRM targeted mass spectrometry. This expansion of measurable Esx-1 proteins allowed us to define statistical rules for assigning novel substrates using phenotypic profiles of known Esx-1 substrates. Using this approach, we identified three additional Esx-1 substrates encoded by the esx-1 region. Our studies begin to address how disruption of specific genes affects several proteins in the Esx-1 complex. Overall, our findings illuminate relationships between Esx-1 proteins and create a framework for the identification of secreted substrates applicable to other protein exporters and pathways. PMID:25106450

  8. Seropositivity ofChlamydophila pneumoniae immunoglobulin G antibody of HIV/AIDS patients in Abuja, Nigeria

    Institute of Scientific and Technical Information of China (English)

    Yakubu Boyi Ngwai; Izebe KS; Ijele IG; Ishaleku D; Inyang US

    2010-01-01

    Objective:To detect IgG antibody toChlamydophila pneumoniae(CP)in sera ofHIV/AIDS patients and provide rationale for inclusion of routine screening for anti-CP antibodies and anti-chlamydial agents in the Nigerian NationalHIV/AIDS Management Plan.Methods: Serum samples from34 consentingHIV/AIDS patients attended a Government-approved Antiretroviral Treatment Facility in Abuja were screened by enzyme-linked immunosorbent assay for anti-CP IgG antibody using ImmunoComb® Chlamydia Bivalent IgG Test kit (Orgenics, Israel).Results:Anti-CP IgG antibody was detected in20 (58.8%)of34 patients tested. The detection rate was higher among the males(8/13; 61.5%) than the females (12/21; 57.1%). Patients of the age group 16-30 years had the highest(7/10; 70%) detection of anti-CP IgG antibody.Conclusions:The result of the present study suggests the presence of anti-CP antibodies in sera of the HIV/AIDS patients, and reinforces the need for routine screening for anti-CPantibodies as a necessary intervention to reduce the burden ofChlamydophila pneumoniae (C. pneumoniae) infections and to reduceHIV-positive morbidity in Nigeria. The outcome of this study also provides justification for the possible inclusion of anti-chlamydial agents in the NationalHIV/AIDS Management Plan to provide prophylaxis against or treat activeC. pneumoniae infections.

  9. Enhancing the Standard of Teaching and Learning in the 21st Century via Qualitative School-Based Supervision in Secondary Schools in Abuja Municipal Area Council (AMAC)

    Science.gov (United States)

    Ebele, Uju F.; Olofu, Paul A.

    2017-01-01

    The study focused on enhancing the standard of teaching and learning in the 21st century via qualitative school-based supervision in secondary schools in Abuja municipal area council. To guide the study, two null hypotheses were formulated. A descriptive survey research design was adopted. The sample of the study constituted of 270 secondary…

  10. Large-scale screening of a targeted Enterococcus faecalis mutant library identifies envelope fitness factors.

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    Lionel Rigottier-Gois

    Full Text Available Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC% gram-positive bacteria. Among those, Enterococcus faecalis is a leading cause of hospital-acquired infections associated with life-threatening issues and increased hospital costs. To better understand the molecular properties of enterococci that may be required for virulence, and that may explain the emergence of these bacteria in nosocomial infections, we performed the first large-scale functional analysis of E. faecalis V583, the first vancomycin-resistant isolate from a human bloodstream infection. E. faecalis V583 is within the high-risk clonal complex 2 group, which comprises mostly isolates derived from hospital infections worldwide. We conducted broad-range screenings of candidate genes likely involved in host adaptation (e.g., colonization and/or virulence. For this purpose, a library was constructed of targeted insertion mutations in 177 genes encoding putative surface or stress-response factors. Individual mutants were subsequently tested for their i resistance to oxidative stress, ii antibiotic resistance, iii resistance to opsonophagocytosis, iv adherence to the human colon carcinoma Caco-2 epithelial cells and v virulence in a surrogate insect model. Our results identified a number of factors that are involved in the interaction between enterococci and their host environments. Their predicted functions highlight the importance of cell envelope glycopolymers in E. faecalis host adaptation. This study provides a valuable genetic database for understanding the steps leading E. faecalis to opportunistic virulence.

  11. A new peptide ligand for targeting human carbonic anhydrase IX, identified through the phage display technology.

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    Vasileios Askoxylakis

    Full Text Available UNLABELLED: Carbonic anhydrase IX (CAIX is a transmembrane enzyme found to be overexpressed in various tumors and associated with tumor hypoxia. Ligands binding this target may be used to visualize hypoxia, tumor manifestation or treat tumors by endoradiotherapy. METHODS: Phage display was performed with a 12 amino acid phage display library by panning against a recombinant extracellular domain of human carbonic anhydrase IX. The identified peptide CaIX-P1 was chemically synthesized and tested in vitro on various cell lines and in vivo in Balb/c nu/nu mice carrying subcutaneously transplanted tumors. Binding, kinetic and competition studies were performed on the CAIX positive human renal cell carcinoma cell line SKRC 52, the CAIX negative human renal cell carcinoma cell line CaKi 2, the human colorectal carcinoma cell line HCT 116 and on human umbilical vein endothelial cells (HUVEC. Organ distribution studies were carried out in mice, carrying SKRC 52 tumors. RNA expression of CAIX in HCT 116 and HUVEC cells was investigated by quantitative real time PCR. RESULTS: In vitro binding experiments of (125I-labeled-CaIX-P1 revealed an increased uptake of the radioligand in the CAIX positive renal cell carcinoma cell line SKRC 52. Binding of the radioligand in the colorectal carcinoma cell line HCT 116 increased with increasing cell density and correlated with the mRNA expression of CAIX. Radioligand uptake was inhibited up to 90% by the unlabeled CaIX-P1 peptide, but not by the negative control peptide octreotide at the same concentration. No binding was demonstrated in CAIX negative CaKi 2 and HUVEC cells. Organ distribution studies revealed a higher accumulation in SKRC 52 tumors than in heart, spleen, liver, muscle, intestinum and brain, but a lower uptake compared to blood and kidney. CONCLUSIONS: These data indicate that CaIX-P1 is a promising candidate for the development of new ligands targeting human carbonic anhydrase IX.

  12. Urban governance and spatial inequality in service delivery: a case study of solid waste management in Abuja, Nigeria.

    Science.gov (United States)

    Adama, Onyanta

    2012-09-01

    Spatial inequality in service delivery is a common feature in African cities. Several factors account for the phenomenon but there is growing attention towards urban governance and the role of the state. Urban governance policies such as privatization serve as key strategies through which the state regulates and (re)produces spatial inequality in service delivery. This study examined how governance practices related to privatization and the regulatory role of the state reinforce spatial inequalities in the delivery of solid waste services in Abuja, Nigeria. It focused primarily on the issue of cost recovery. Privatization became a major focus in Abuja in 2003 when the government launched a pilot scheme. Although it has brought improvements in service delivery, privatization has also increased the gap in the quality of services delivered in different parts of the city. Drawing on empirical data, the study revealed that little sensitivity to income and affordability, and to income differentials between neighbourhoods in the fixing of user charges and in the choice of the billing method is contributing to spatial inequalities in service delivery. Furthermore, the study suggests that these practices are linked to a broader issue, a failure of the government to see the people as partners. It therefore calls for more inclusive governance especially in decision-making processes. The study also emphasizes the need for a policy document on solid waste management, as this would encourage a critical assessment of vital issues including how privatization is to be funded, especially in low-income areas.

  13. COINCIDENCES BETWEEN ELECTRONS AND TARGET IONS TO IDENTIFY CAPTURE CHANNELS IN COLLISIONS OF MULTIPLY CHARGED IONS ON GAS TARGETS

    NARCIS (Netherlands)

    POSTHUMUS, JH; MORGENSTERN, R

    1992-01-01

    We have investigated multielectron capture processes in collisions of Ar9+ on Ar by measuring the resulting Auger electrons in coincidence with charge-state-analyzed target ions. In this way it was possible to reconstruct partial electron energy spectra, each corresponding to a particular number of

  14. Comparative analysis of prevalence of intimate partner violence against women in military and civilian communities in Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Chimah CU

    2015-03-01

    Full Text Available Carol Uzoamaka Chimah,1 Prosper Obunikem Uche Adogu,2 Kofoworola Odeyemi,3 Amobi Linus Ilika4 1Medical Department, Ministry of Defence Headquarters, Abuja, Nigeria; 2Department of Community Medicine and PHC, Nnamdi Azikiwe University/Nnamdi Azikiwe University Teaching Hospital, Nnewi, Nigeria; 3Department of Community Health, University of Lagos, Lagos, Nigeria Introduction: Intimate partner violence (IPV occurs across the world, in various cultures, and affects people across societies irrespective of economic status or gender. Most data on IPV before World Health Organization multicountry study (WHOMCS usually came from sources other than the military. Result of this study will contribute to the existing body of knowledge and may serve as a baseline for future studies in military populations. This study compares the prevalence of the different types of IPV against women in military and civilian communities in Abuja, Nigeria.Methods: Using a multistage sampling technique, 260 women who had intimate male partners were selected from military and civilian communities of Abuja. Collected data on personal characteristics and different types of IPV experienced were analyzed to demonstrate comparison of the association between the different forms of IPV and the respondents’ sociodemographic and partner characteristics in the two study populations using percentages and Χ-square statistics, and P-value was assumed to be significant at ≤0.05.Results: The prevalence of the four major types of IPV was higher among the military respondents than among civilians: controlling behavior, 37.1% versus 29.1%; emotional/psychological abuse, 42.4% versus 13.4%; physical abuse, 19.7% versus 5.9%, and sexual abuse, 9.2% versus 8.8%. Significantly more respondents from the military population (59 [45.4%] compared to civilians (21 [19.4%] were prevented by their partners from seeing their friends (P=0.000. The situation is reversed with regard to permission to seek

  15. Novel snail1 target proteins in human colon cancer identified by proteomic analysis.

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    María Jesús Larriba

    Full Text Available BACKGROUND: The transcription factor Snail1 induces epithelial-to-mesenchymal transition (EMT, a process responsible for the acquisition of invasiveness during tumorigenesis. Several transcriptomic studies have reported Snail1-regulated genes in different cell types, many of them involved in cell adhesion. However, only a few studies have used proteomics as a tool for the characterization of proteins mediating EMT. METHODOLOGY/PRINCIPAL FINDINGS: We identified by proteomic analysis using 2D-DIGE electrophoresis combined with MALDI-TOF-TOF and ESI-linear ion trap mass spectrometry a number of proteins with variable functions whose expression is modulated by Snail1 in SW480-ADH human colon cancer cells. Validation was performed by Western blot and immunofluorescence analyses. Snail1 repressed several members of the 14-3-3 family of phosphoserine/phosphothreonine binding proteins and also the expression of the Proliferation-associated protein 2G4 (PA2G4 that was mainly localized at the nuclear Cajal bodies. In contrast, the expression of two proteins involved in RNA processing, the Cleavage and polyadenylation specificity factor subunit 6 (CPSF6 and the Splicing factor proline/glutamine-rich (SFPQ, was higher in Snail1-expressing cells than in controls. The regulation of 14-3-3epsilon, 14-3-3tau, 14-3-3zeta and PA2G4 by Snail1 was reproduced in HT29 colon cancer cells. In addition, we found an inverse correlation between 14-3-3sigma and Snail1 expression in human colorectal tumors. CONCLUSIONS/SIGNIFICANCE: We have identified a set of novel Snail1 target proteins in colon cancer that expand the cellular processes affected by Snail1 and thus its relevance for cell function and phenotype.

  16. Hospital to Post-Acute Care Facility Transfers: Identifying Targets for Information Exchange Quality Improvement.

    Science.gov (United States)

    Jones, Christine D; Cumbler, Ethan; Honigman, Benjamin; Burke, Robert E; Boxer, Rebecca S; Levy, Cari; Coleman, Eric A; Wald, Heidi L

    2017-01-01

    Information exchange is critical to high-quality care transitions from hospitals to post-acute care (PAC) facilities. We conducted a survey to evaluate the completeness and timeliness of information transfer and communication between a tertiary-care academic hospital and its related PAC facilities. This was a cross-sectional Web-based 36-question survey of 110 PAC clinicians and staff representing 31 PAC facilities conducted between October and December 2013. We received responses from 71 of 110 individuals representing 29 of 31 facilities (65% and 94% response rates). We collapsed 4-point Likert responses into dichotomous variables to reflect completeness (sufficient vs insufficient) and timeliness (timely vs not timely) for information transfer and communication. Among respondents, 32% reported insufficient information about discharge medical conditions and management plan, and 83% reported at least occasionally encountering problems directly related to inadequate information from the hospital. Hospital clinician contact information was the most common insufficient domain. With respect to timeliness, 86% of respondents desired receipt of a discharge summary on or before the day of discharge, but only 58% reported receiving the summary within this time frame. Through free-text responses, several participants expressed the need for paper prescriptions for controlled pain medications to be sent with patients at the time of transfer. Staff and clinicians at PAC facilities perceive substantial deficits in content and timeliness of information exchange between the hospital and facilities. Such deficits are particularly relevant in the context of the increasing prevalence of bundled payments for care across settings as well as forthcoming readmissions penalties for PAC facilities. Targets identified for quality improvement include structuring discharge summary information to include information identified as deficient by respondents, completion of discharge summaries

  17. Getting to zero the biomedical way in Africa: outcomes of deliberation at the 2013 Biomedical HIV Prevention Forum in Abuja, Nigeria.

    Science.gov (United States)

    Folayan, Morenike Oluwatoyin; Gottemoeller, Megan; Mburu, Rosemary; Brown, Brandon

    2014-01-01

    Over the last few decades, biomedical HIV prevention research had engaged multiple African stakeholders. There have however been few platforms to enable regional stakeholders to engage with one another. In partnership with the World AIDS Campaign International, the Institute of Public Health of Obafemi Awolowo University, and the National Agency for the Control of AIDS in Nigeria, the New HIV Vaccine and Microbicide Advocacy Society hosted a forum on biomedical HIV prevention research in Africa. Stakeholders' present explored evidences related to biomedical HIV prevention research and development in Africa, and made recommendations to inform policy, guidelines and future research agenda. The BHPF hosted 342 participants. Topics discussed included the use of antiretrovirals for HIV prevention, considerations for biomedical HIV prevention among key populations; HIV vaccine development; HIV cure; community and civil society engagement; and ethical considerations in implementation of biomedical HIV prevention research. Participants identified challenges for implementation of proven efficacious interventions and discovery of other new prevention options for Africa. Concerns raised included limited funding by African governments, lack of cohesive advocacy and policy agenda for biomedical HIV prevention research and development by Africa, varied ethical practices, and limited support to communities' capacity to actively engaged with clinical trial conducts. Participants recommended that the African Government implement the Abuja +12 declaration; the civil society build stronger partnerships with diverse stakeholders, and develop a coherent advocacy agenda that also enhances community research literacy; and researchers and sponsors of trials on the African continent establish a process for determining appropriate standards for trial conduct on the continent. By highlighting key considerations for biomedical HIV prevention research and development in Africa, the forum has

  18. Novel target for high-risk neuroblastoma identified in pre-clinical research | Center for Cancer Research

    Science.gov (United States)

    Pre-clinical research by investigators at the Center for Cancer Research and their colleagues have identified a number of novel epigenetic targets for high-risk neuroblastoma and validated a promising new targeted inhibitor in pre-clinical models.  Read more...

  19. A non-target approach to identify disinfection byproducts of structurally similar sulfonamide antibiotics.

    Science.gov (United States)

    Wang, Mian; Helbling, Damian E

    2016-10-01

    There is growing concern over the formation of new types of disinfection byproducts (DBPs) from pharmaceuticals and other emerging contaminants during drinking water production. Free chlorine is a widely used disinfectant that reacts non-selectively with organic molecules to form a variety of byproducts. In this research, we aimed to investigate the DBPs formed from three structurally similar sulfonamide antibiotics (sulfamethoxazole, sulfathiazole, and sulfadimethoxine) to determine how chemical structure influences the types of chlorination reactions observed. We conducted free chlorination experiments and developed a non-target approach to extract masses from the experimental dataset that represent the masses of candidate DBPs. Structures were assigned to the candidate DBPs based on analytical data and knowledge of chlorine chemistry. Confidence levels were assigned to each proposed structure according to conventions in the field. In total, 11, 12, and 15 DBP structures were proposed for sulfamethoxazole, sulfathiazole, and sulfadimethoxine, respectively. The structures of the products suggest a variety of reaction types including chlorine substitution, SC cleavage, SN hydrolysis, desulfonation, oxidation/hydroxylation, and conjugation reactions. Some reaction types were common to all of the sulfonamide antibiotics, but unique reaction types were also observed for each sulfonamide antibiotic suggesting that selective prediction of DBP structures of other sulfonamide antibiotics based on chemical structure is unlikely to be possible based on these data alone. This research offers an approach to comprehensively identify DBPs of organic molecules and fills in much needed data on the formation of specific DBPs from three environmentally relevant sulfonamide antibiotics.

  20. Microarray analysis of human monocytes infected with Francisella tularensis identifies new targets of host response subversion.

    Directory of Open Access Journals (Sweden)

    Jonathan P Butchar

    Full Text Available Francisella tularensis is a gram-negative facultative bacterium that causes the disease tularemia, even upon exposure to low numbers of bacteria. One critical characteristic of Francisella is its ability to dampen or subvert the host immune response. In order to help understand the mechanisms by which this occurs, we performed Affymetrix microarray analysis on transcripts from blood monocytes infected with the virulent Type A Schu S4 strain. Results showed that expression of several host response genes were reduced such as those associated with interferon signaling, Toll-like receptor signaling, autophagy and phagocytosis. When compared to microarrays from monocytes infected with the less virulent F. tularensis subsp. novicida, we found qualitative differences and also a general pattern of quantitatively reduced pro-inflammatory signaling pathway genes in the Schu S4 strain. Notably, the PI3K/Akt1 pathway appeared specifically down-regulated following Schu S4 infection and a concomitantly lower cytokine response was observed. This study identifies several new factors potentially important in host cell subversion by the virulent Type A F. tularensis that may serve as novel targets for drug discovery.

  1. Identifying initial molecular targets of PDT: protein and lipid oxidation products

    Science.gov (United States)

    Oleinick, Nancy L.; Kim, Junhwan; Rodriguez, Myriam E.; Xue, Liang-yan; Kenney, Malcolm E.; Anderson, Vernon E.

    2009-06-01

    Photodynamic Therapy (PDT) generates singlet oxygen (1O2) which oxidizes biomolecules in the immediate vicinity of its formation. The phthalocyanine photosensitizer Pc 4 localizes to mitochondria and endoplasmic reticulum, and the primary targets of Pc 4-PDT are expected to be lipids and proteins of those membranes. The initial damage then causes apoptosis in cancer cells via the release of cytochrome c (Cyt-c) from mitochondria into the cytosol, followed by the activation of caspases. That damage also triggers the induction of autophagy, an attempt by the cells to eliminate damaged organelles, or when damage is too extensive, to promote cell death. Cyt-c is bound to the cytosolic side of the mitochondrial inner membrane through association with cardiolipin (CL), a phospholipid containing four unsaturated fatty acids and thus easily oxidized by 1O2 or by other oxidizing agents. Increasing evidence suggests that oxidation of CL loosens its association with Cyt-c, and that the peroxidase activity of Cyt-c can oxidize CL. In earlier studies of Cyt-c in homogeneous medium by MALDI-TOF-MS and LC-ESI-MS, we showed that 1O2 generated by Pc 4-PDT oxidized histidine, methionine, tryptophan, and unexpectedly phenylalanine but not tyrosine. Most of the oxidation products were known to be formed by other oxidizing agents, such as hydroxyl radical, superoxide radical anion, and peroxynitrite. However, two products of histidine were unique to 1O2 and may be useful for reporting the action of 1O2 in cells and tissues. These products, as well as CL oxidation products, have now been identified in liposomes and mitochondria after Pc 4-PDT. In mitochondria, the PDT dose-dependent oxidations can be related to specific changes in mitochondrial function, Bcl-2 photodamage, and Cyt-c release. Thus, the role of PDT-generated 1O2 in oxidizing Cyt-c and CL and the interplay between protein and lipid targets may be highly relevant to understanding one mechanism for cell killing by PDT.

  2. Using a blackboard architecture or expert system to identify obfuscated targets from symptoms

    Science.gov (United States)

    Straub, Jeremy

    2015-05-01

    A variety of techniques exist for enhancing or inferring the existence and characteristics of an obscured or partially concealed target. Targets, however, may be completely blocked from view, presenting nothing to enhance and no image area to extend inferentially. Despite the difficulty, concealed (particularly intentionally) targets may be the most important to detect. This paper proposes a technique for using a Blackboard Architecture or Expert system to infer a target's existence from symptoms (maneuvers of other units, water and soil deformation, etc.) and discusses the differences between the two approaches (Blackboard Architecture and expert system) for doing so.

  3. Transcriptome Profiling Identifies Ribosome Biogenesis as a Target of Alcohol Teratogenicity and Vulnerability during Early Embryogenesis

    Science.gov (United States)

    Berres, Mark E.; Garic, Ana; Flentke, George R.; Smith, Susan M.

    2017-01-01

    Fetal alcohol spectrum disorder (FASD) is a leading cause of neurodevelopmental disability. Individuals with FASD may exhibit a characteristic facial appearance that has diagnostic utility. The mechanism by which alcohol disrupts craniofacial development is incompletely understood, as are the genetic factors that can modify individual alcohol vulnerability. Using an established avian model, we characterized the cranial transcriptome in response to alcohol to inform the mechanism underlying these cells’ vulnerability. Gallus gallus embryos having 3–6 somites were exposed to 52 mM alcohol and the cranial transcriptomes were sequenced thereafter. A total of 3422 genes had significantly differential expression. The KEGG pathways with the greatest enrichment of differentially expressed gene clusters were Ribosome (P = 1.2 x 10−17, 67 genes), Oxidative Phosphorylation (P = 4.8 x 10−12, 60 genes), RNA Polymerase (P = 2.2 x 10−3, 15 genes) and Spliceosome (P = 2.6 x 10−2, 39 genes). The preponderance of transcripts in these pathways were repressed in response to alcohol. These same gene clusters also had the greatest altered representation in our previous comparison of neural crest populations having differential vulnerability to alcohol-induced apoptosis. Comparison of differentially expressed genes in alcohol-exposed (3422) and untreated, alcohol-vulnerable (1201) transcriptomes identified 525 overlapping genes of which 257 have the same direction of transcriptional change. These included 36 ribosomal, 25 oxidative phosphorylation and 7 spliceosome genes. Using a functional approach in zebrafish, partial knockdown of ribosomal proteins zrpl11, zrpl5a, and zrps3a individually heightened vulnerability to alcohol-induced craniofacial deficits and increased apoptosis. In humans, haploinsufficiency of several of the identified ribosomal proteins are causative in craniofacial dysmorphologies such as Treacher Collins Syndrome and Diamond-Blackfan Anemia. This work

  4. Network analysis of translocated Takahe populations to identify disease surveillance targets.

    Science.gov (United States)

    Grange, Zoë L; VAN Andel, Mary; French, Nigel P; Gartrell, Brett D

    2014-04-01

    network in 2011. Likewise, the wild Murchison Mountains population was consistently the sink of the network. Other nodes, such as the offshore islands and the wildlife hospital, varied in importance over time. Common network descriptors and measures of centrality identified key locations for targeting disease surveillance. The visual representation of movements of animals in a population that this technique provides can aid decision makers when they evaluate translocation proposals or attempt to control a disease outbreak. © 2014 Society for Conservation Biology.

  5. Targeted capture enrichment and sequencing identifies extensive nucleotide variation in the turkey MHC-B.

    Science.gov (United States)

    Reed, Kent M; Mendoza, Kristelle M; Settlage, Robert E

    2016-03-01

    Variation in the major histocompatibility complex (MHC) is increasingly associated with disease susceptibility and resistance in avian species of agricultural importance. This variation includes sequence polymorphisms but also structural differences (gene rearrangement) and copy number variation (CNV). The MHC has now been described for multiple galliform species including the best defined assemblies of the chicken (Gallus gallus) and domestic turkey (Meleagris gallopavo). Using this sequence resource, this study applied high-throughput sequencing to investigate MHC variation in turkeys of North America (NA turkeys). An MHC-specific SureSelect (Agilent) capture array was developed, and libraries were created for 14 turkeys representing domestic (commercial bred), heritage breed, and wild turkeys. In addition, a representative of the Ocellated turkey (M. ocellata) and chicken (G. gallus) was included to test cross-species applicability of the capture array allowing for identification of new species-specific polymorphisms. Libraries were hybridized to ∼12 K cRNA baits and the resulting pools were sequenced. On average, 98% of processed reads mapped to the turkey whole genome sequence and 53% to the MHC target. In addition to the MHC, capture hybridization recovered sequences corresponding to other MHC regions. Sequence alignment and de novo assembly indicated the presence of several additional BG genes in the turkey with evidence for CNV. Variant detection identified an average of 2245 polymorphisms per individual for the NA turkeys, 3012 for the Ocellated turkey, and 462 variants in the chicken (RJF-256). This study provides an extensive sequence resource for examining MHC variation and its relation to health of this agriculturally important group of birds.

  6. Efficient Isothermal Titration Calorimetry Technique Identifies Direct Interaction of Small Molecule Inhibitors with the Target Protein.

    Science.gov (United States)

    Gal, Maayan; Bloch, Itai; Shechter, Nelia; Romanenko, Olga; Shir, Ofer M

    2016-01-01

    Protein-protein interactions (PPI) play a critical role in regulating many cellular processes. Finding novel PPI inhibitors that interfere with specific binding of two proteins is considered a great challenge, mainly due to the complexity involved in characterizing multi-molecular systems and limited understanding of the physical principles governing PPIs. Here we show that the combination of virtual screening techniques, which are capable of filtering a large library of potential small molecule inhibitors, and a unique secondary screening by isothermal titration calorimetry, a label-free method capable of observing direct interactions, is an efficient tool for finding such an inhibitor. In this study we applied this strategy in a search for a small molecule capable of interfering with the interaction of the tumor-suppressor p53 and the E3-ligase MDM2. We virtually screened a library of 15 million small molecules that were filtered to a final set of 80 virtual hits. Our in vitro experimental assay, designed to validate the activity of mixtures of compounds by isothermal titration calorimetry, was used to identify an active molecule against MDM2. At the end of the process the small molecule (4S,7R)-4-(4-chlorophenyl)-5-hydroxy-2,7-dimethyl-N-(6-methylpyridin-2-yl)-4,6,7,8 tetrahydrIoquinoline-3-carboxamide was found to bind MDM2 with a dissociation constant of ~2 µM. Following the identification of this single bioactive compound, spectroscopic measurements were used to further characterize the interaction of the small molecule with the target protein. 2D NMR spectroscopy was used to map the binding region of the small molecule, and fluorescence polarization measurement confirmed that it indeed competes with p53.

  7. Comparative analysis of prevalence of intimate partner violence against women in military and civilian communities in Abuja, Nigeria.

    Science.gov (United States)

    Chimah, Carol Uzoamaka; Adogu, Prosper Obunikem Uche; Odeyemi, Kofoworola; Ilika, Amobi Linus

    2015-01-01

    Intimate partner violence (IPV) occurs across the world, in various cultures, and affects people across societies irrespective of economic status or gender. Most data on IPV before World Health Organization multicountry study (WHOMCS) usually came from sources other than the military. Result of this study will contribute to the existing body of knowledge and may serve as a baseline for future studies in military populations. This study compares the prevalence of the different types of IPV against women in military and civilian communities in Abuja, Nigeria. Using a multistage sampling technique, 260 women who had intimate male partners were selected from military and civilian communities of Abuja. Collected data on personal characteristics and different types of IPV experienced were analyzed to demonstrate comparison of the association between the different forms of IPV and the respondents' sociodemographic and partner characteristics in the two study populations using percentages and χ-square statistics, and P-value was assumed to be significant at ≤0.05. The prevalence of the four major types of IPV was higher among the military respondents than among civilians: controlling behavior, 37.1% versus 29.1%; emotional/psychological abuse, 42.4% versus 13.4%; physical abuse, 19.7% versus 5.9%, and sexual abuse, 9.2% versus 8.8%. Significantly more respondents from the military population (59 [45.4%]) compared to civilians (21 [19.4%]) were prevented by their partners from seeing their friends (P=0.000). The situation is reversed with regard to permission to seek health care for self, with civilians reporting a significantly higher prevalence (35 [32.4%]) than did military respondents (20 [15.4%]) (P=0.002). The military respondents were clearly at a higher risk of experiencing all the variants of emotional violence than the civilians (P=0.00). The commonest form of physical violence against women was "being slapped or having something thrown at them, that could hurt

  8. COSMID: A Web-based Tool for Identifying and Validating CRISPR/Cas Off-target Sites

    Directory of Open Access Journals (Sweden)

    Thomas J Cradick

    2014-01-01

    Full Text Available Precise genome editing using engineered nucleases can significantly facilitate biological studies and disease treatment. In particular, clustered regularly interspaced short palindromic repeats (CRISPR with CRISPR-associated (Cas proteins are a potentially powerful tool for modifying a genome by targeted cleavage of DNA sequences complementary to designed guide strand RNAs. Although CRISPR/Cas systems can have on-target cleavage rates close to the transfection rates, they may also have relatively high off-target cleavage at similar genomic sites that contain one or more base pair mismatches, and insertions or deletions relative to the guide strand. We have developed a bioinformatics-based tool, COSMID (CRISPR Off-target Sites with Mismatches, Insertions, and Deletions that searches genomes for potential off-target sites (http://crispr.bme.gatech.edu. Based on the user-supplied guide strand and input parameters, COSMID identifies potential off-target sites with the specified number of mismatched bases and insertions or deletions when compared with the guide strand. For each site, amplification primers optimal for the chosen application are also given as output. This ranked-list of potential off-target sites assists the choice and evaluation of intended target sites, thus helping the design of CRISPR/Cas systems with minimal off-target effects, as well as the identification and quantification of CRISPR/Cas induced off-target cleavage in cells.

  9. An In Vivo Method to Identify microRNA Targets Not Predicted by Computation Algorithms: p21 Targeting by miR-92a in Cancer.

    Science.gov (United States)

    Su, Xiaoping; Wang, Huaming; Ge, Wei; Yang, Mingjin; Hou, Jin; Chen, Taoyong; Li, Nan; Cao, Xuetao

    2015-07-15

    microRNA (miRNA) dysregulation is involved in the development and progression of various human cancers, including hepatocellular carcinoma (HCC). However, how to identify the miRNAs targeting specific mRNA in cells is a significant challenge because of the interaction complexity and the limited knowledge of rules governing these processes. Some miRNAs are not predictable by current computer algorithms available. Here, using p21 mRNA as target, we established a new method, called miRNA in vivo precipitation (miRIP), to identify which kind of miRNAs can actually bind to the specific mRNA in cells. Several unpredictable miRNAs that bound p21 mRNA in HepG2 and PC-3 cells were identified by the miRIP method. Among these miRNAs identified by miRIP, miR-92a was found and confirmed to interact robustly with p21 mRNA, both in HepG2 and PC-3 cells. miR-92a was found to be remarkably increased in HCC tissues, and higher expression of miR-92a significantly correlated with lower expression of p21, which is related to poor survival of HCC patients. Moreover, inhibition of miR-92a could significantly suppress HCC growth in vitro and in vivo by upregulating p21. Together, miR-92a, which is identified by miRIP, is functionally shown to be associated with HCC growth as an oncogenic miRNA by inhibiting expression of targeting gene p21. In addition, several unpredictable miRNAs that target STAT3 mRNA were also identified by the miRIP method in HepG2 cells. Our results demonstrated that the miRIP approach can effectively identify the unpredictable but intracellular existing miRNAs that target specific mRNA in vivo.

  10. Modes of targets in water excited and identified using radiation pressure of modulated focused ultrasound

    Science.gov (United States)

    Daniel, Timothy; Fortuner, Auberry; Abawi, Ahmad; Kirsteins, Ivars; Marston, Philip

    2016-11-01

    The modulated radiation pressure (MRP) of ultrasound has been widely used to selectively excite low frequency modes of fluid objects. We previously used MRP to excite less compliant metallic object in water including the low frequency modes of a circular metal plate in water. A larger focused ultrasonic transducer allows us to drive modes of larger more-realistic targets. In our experiments solid targets are suspended by strings or supported on sand and the modulated ultrasound is focused on the target's surface. Target sound emissions were recorded and a laser vibrometer was used to measure the surface velocity of the target to give the magnitude of the target response. The source transducer was driven with a doublesideband suppressed carrier voltage as in. By varying the modulation frequency and monitoring target response, resonant frequencies can be measured and compared to finite element models. We also demonstrate the radiation torque of a focused first-order acoustic vortex beam associated with power absorption in the Stokes layer adjacent to a sphere. Funded by ONR.

  11. Potential of phage-displayed peptide library technology to identify functional targeting peptides

    Science.gov (United States)

    Krumpe, Lauren RH; Mori, Toshiyuki

    2010-01-01

    Combinatorial peptide library technology is a valuable resource for drug discovery and development. Several peptide drugs developed through phage-displayed peptide library technology are presently in clinical trials and the authors envision that phage-displayed peptide library technology will assist in the discovery and development of many more. This review attempts to compile and summarize recent literature on targeting peptides developed through peptide library technology, with special emphasis on novel peptides with targeting capacity evaluated in vivo. PMID:20150977

  12. Reverse screening approach to identify potential anti-cancer targets of dipyridamole

    Science.gov (United States)

    Ge, Shu-Min; Zhan, Dong-Ling; Zhang, Shu-Hua; Song, Li-Qiang; Han, Wei-Wei

    2016-01-01

    Dipyridamole (DIP) inhibits thrombus formation when given chronically, and causes vasodilation over a short time. To date, DIP can increase the anticancer drugs (5-fluorouracil, methotrexate, piperidine, vincristine) concentration in cancer cells and hence enhance the efficacy of treatment cancer. The inhibition of DIP may result in increased 5-fluorouracil efficacy and diminish the drug side effects. But the actual molecular targets remain unknown. In this study, reverse protein-ligands docking, and quantum mechanics were used to search for the potential molecular targets of DIP. The quantum mechanics calculation was performed by using Gaussian 03 program package. Reverse pharmacophore mapping was used to search for potential molecular target candidates for a given small molecule. The docking study was used for exploring the potential anti-cancer targets of dipyridamole. The two predicted binders with the statistically significant prediction are dihydropyrimidine dehydrogenase (DPD) (PDB Id: 1GTE) and human spindle checkpoint kinase Bub1 (PDB Id: 3E7E). Structure analysis suggests that electrostatic interaction and hydrogen bonding play an important role in their binding process. The strong functional linkage of DIP and 5FU supports our prediction. In conclusion, these results generate a tractable set of anticancer proteins. The exploration of polypharmacology will provide us new opportunities in treating systematic diseases, such as the cancers. The results would generate a tractable set of anticancer target proteins for future experimental validations. PMID:28077994

  13. In Vivo Selection Against Human Colorectal Cancer Xenografts Identifies an Aptamer That Targets RNA Helicase Protein DHX9

    Directory of Open Access Journals (Sweden)

    Jing Mi

    2016-01-01

    Full Text Available The ability to selectively target disease-related tissues with molecules is critical to the design of effective therapeutic and diagnostic reagents. Recognizing the differences between the in vivo environment and in vitro conditions, we employed an in vivo selection strategy to identify RNA aptamers (targeting motifs that could localize to tumor in situ. One of the selected molecules is an aptamer that binds to the protein DHX9, an RNA helicase that is known to be upregulated in colorectal cancer. Upon systemic administration, the aptamer preferentially localized to the nucleus of cancer cells in vivo and thus has the potential to be used for targeted delivery.

  14. Knowledge of breast cancer and practice of breast self examination among female senior secondary school students in Abuja, Nigeria.

    Science.gov (United States)

    Isara, A R; Ojedokun, C I

    2011-12-01

    Breast cancer is a public health problem that is increasing throughout the world especially in developing countries. The study was aimed at assessing the knowledge of breast cancer and practice of breast self examination (BSE) among female senior secondary school students in the municipal council area of Abuja, Nigeria. This descriptive cross sectional study was carried out among female senior secondary school students from selected schools in the municipal area council of Abuja. The tool for data collection was a structured self administered questionnaire. Data were analysed using SPSS version 16.0. Two hundred and eighty-seven students participated in the study. Their mean age was 16.5 +/- 1.4 years. A greater proportion of respondents 163 (56.8%) had poor knowledge of breast cancer while 217 (75.6%) had poor knowledge of BSE. Only 114 (39.7%) of the respondents knew that being a female was a risk factor for breast cancer and the least known risk factors were obesity and aging. The major source of information for breast cancer and BSE among the respondents was the mass media. Only 29 (10.1%) of respondents had practiced BSE. Knowledge of BSE was significantly associated with BSE practice. This study revealed that female secondary school students have poor knowledge of breast cancer. A good proportion of them knew that BSE could be used as a screening method for breast cancer but only few had practiced BSE. There is need for adequate health education on breast cancer and BSE among adolescent females in Nigeria.

  15. Utilizing Chemical Genomics to Identify Cytochrome b as a Novel Drug Target for Chagas Disease.

    Directory of Open Access Journals (Sweden)

    Shilpi Khare

    2015-07-01

    Full Text Available Unbiased phenotypic screens enable identification of small molecules that inhibit pathogen growth by unanticipated mechanisms. These small molecules can be used as starting points for drug discovery programs that target such mechanisms. A major challenge of the approach is the identification of the cellular targets. Here we report GNF7686, a small molecule inhibitor of Trypanosoma cruzi, the causative agent of Chagas disease, and identification of cytochrome b as its target. Following discovery of GNF7686 in a parasite growth inhibition high throughput screen, we were able to evolve a GNF7686-resistant culture of T. cruzi epimastigotes. Clones from this culture bore a mutation coding for a substitution of leucine by phenylalanine at amino acid position 197 in cytochrome b. Cytochrome b is a component of complex III (cytochrome bc1 in the mitochondrial electron transport chain and catalyzes the transfer of electrons from ubiquinol to cytochrome c by a mechanism that utilizes two distinct catalytic sites, QN and QP. The L197F mutation is located in the QN site and confers resistance to GNF7686 in both parasite cell growth and biochemical cytochrome b assays. Additionally, the mutant cytochrome b confers resistance to antimycin A, another QN site inhibitor, but not to strobilurin or myxothiazol, which target the QP site. GNF7686 represents a promising starting point for Chagas disease drug discovery as it potently inhibits growth of intracellular T. cruzi amastigotes with a half maximal effective concentration (EC50 of 0.15 µM, and is highly specific for T. cruzi cytochrome b. No effect on the mammalian respiratory chain or mammalian cell proliferation was observed with up to 25 µM of GNF7686. Our approach, which combines T. cruzi chemical genetics with biochemical target validation, can be broadly applied to the discovery of additional novel drug targets and drug leads for Chagas disease.

  16. Identifying members of the domain Archaea with rRNA-targeted oligonucleotide probes.

    Science.gov (United States)

    Burggraf, S; Mayer, T; Amann, R; Schadhauser, S; Woese, C R; Stetter, K O

    1994-09-01

    Two 16S rRNA-targeted oligonucleotide probes were designed for the archaeal kingdoms Euryachaeota and Crenarchaeota. Probe specificities were evaluated by nonradioactive dot blot hybridization against selected reference organisms. The successful application of fluorescent-probe derivatives for whole-cell hybridization required organism-specific optimizations of fixation and hybridization conditions to assure probe penetration and morphological integrity of the cells. The probes allowed preliminary grouping of three new hyperthermophilic isolates. Together with other group-specific rRNA-targeted oligonucleotide probes, these probes will facilitate rapid in situ monitoring of the populations present in hydrothermal systems and support cultivation attempts.

  17. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

    NARCIS (Netherlands)

    Rigottier-Gois, Lionel; Alberti, Adriana; Houel, Armel; Taly, Jean-François; Palcy, Philippe; Manson, Janet; Pinto, Daniela; Matos, Renata C.; Carrilero, Laura; Montero, Natalia; Tariq, Muhammad; Karsens, Harma; Repp, Christian; Kropec, Andrea; Budin-Verneuil, Aurélie; Benachour, Abdellah; Sauvageot, Nicolas; Bizzini, Alain; Gilmore, Michael S.; Bessières, Philippe; Kok, Jan; Huebner, Johannes; Lopes, Fatima; Gonzalez-Zorn, Bruno; Hartke, Axel; Serror, Pascale

    2011-01-01

    Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC%

  18. A high throughput experimental approach to identify miRNA targets in human cells

    NARCIS (Netherlands)

    Tan, Lu Ping; Seinen, Erwin; Duns, Gerben; de Jong, Debora; Sibon, Ody C. M.; Poppema, Sibrand; Kroesen, Bart-Jan; Kok, Klaas; van den Berg, Anke

    2009-01-01

    The study of human microRNAs is seriously hampered by the lack of proper tools allowing genome-wide identification of miRNA targets. We performed Ribonucleoprotein ImmunoPrecipitation-gene Chip (RIP-Chip) using antibodies against wild-type human Ago2 in untreated Hodgkin lymphoma (HL) cell lines.

  19. Candidiasis drug discovery and development: new approaches targeting virulence for discovering and identifying new drugs

    Science.gov (United States)

    Pierce, Christopher G.; Lopez-Ribot, Jose L.

    2014-01-01

    Introduction Targeting pathogenetic mechanisms rather than essential processes represents a very attractive alternative for the development of new antibiotics. This may be particularly important in the case of antimycotics, due to the urgent need for novel antifungal drugs and the paucity of selective fungal targets. The opportunistic pathogenic fungus Candida albicans is the main etiological agent of candidiasis, the most common human fungal infection. These infections carry unacceptably high mortality rates, a clear reflection of the many shortcomings of current antifungal therapy, including the limited armamentarium of antifungal agents, their toxicity, and the emergence of resistance. Moreover the antifungal pipeline is mostly dry. Areas covered This review covers some of the most recent progress towards understanding C. albicans pathogenetic processes and how to harness this information for the development of anti-virulence agents. The two principal areas covered are filamentation and biofilm formation, as C. albicans pathogenicity is intimately linked to its ability to undergo morphogenetic conversions between yeast and filamentous morphologies and to its ability to form biofilms. Expert opinion We argue that filamentation and biofilm formation represent high value targets, yet clinically unexploited, for the development of novel anti-virulence approaches against candidiasis. Although this has proved a difficult task despite increasing understanding at the molecular level of C. albicans virulence, we highlight new opportunities and prospects for antifungal drug development targeting these two important biological processes. PMID:23738751

  20. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Science.gov (United States)

    Whiteaker, Jeffrey R; Halusa, Goran N; Hoofnagle, Andrew N; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John A; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J; Meyer, Matthew R.; Mesri, Mehdi; Abbatiello, Susan E; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri R; Ellis, Matthew J. C.; Fenyö, David; Hiltke, Tara; Ketchum, Karen A.; Kinsinger, Chris; Kuhn, Eric; Liebler, Daniel C.; Lin, De; Liu, Tao; Loss, Michael; MacCoss, Michael J; Qian, Wei-Jun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly V; Scott, Mitchell G; Smith, Richard D.; Thomas, Stefani; Townsend, R. Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G

    2016-01-01

    Summary The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories. PMID:26867747

  1. Large-Scale Screening of a Targeted Enterococcus faecalis Mutant Library Identifies Envelope Fitness Factors

    NARCIS (Netherlands)

    Rigottier-Gois, Lionel; Alberti, Adriana; Houel, Armel; Taly, Jean-François; Palcy, Philippe; Manson, Janet; Pinto, Daniela; Matos, Renata C.; Carrilero, Laura; Montero, Natalia; Tariq, Muhammad; Karsens, Harma; Repp, Christian; Kropec, Andrea; Budin-Verneuil, Aurélie; Benachour, Abdellah; Sauvageot, Nicolas; Bizzini, Alain; Gilmore, Michael S.; Bessières, Philippe; Kok, Jan; Huebner, Johannes; Lopes, Fatima; Gonzalez-Zorn, Bruno; Hartke, Axel; Serror, Pascale

    2011-01-01

    Spread of antibiotic resistance among bacteria responsible for nosocomial and community-acquired infections urges for novel therapeutic or prophylactic targets and for innovative pathogen-specific antibacterial compounds. Major challenges are posed by opportunistic pathogens belonging to the low GC%

  2. Using the CPTAC Assay Portal to identify and implement highly characterized targeted proteomics assays

    Energy Technology Data Exchange (ETDEWEB)

    Whiteaker, Jeffrey R.; Halusa, Goran; Hoofnagle, Andrew N.; Sharma, Vagisha; MacLean, Brendan; Yan, Ping; Wrobel, John; Kennedy, Jacob; Mani, DR; Zimmerman, Lisa J.; Meyer, Matthew R.; Mesri, Mehdi; Boja, Emily; Carr, Steven A.; Chan, Daniel W.; Chen, Xian; Chen, Jing; Davies, Sherri; Ellis, Matthew; Fenyo, David; Hiltket, Tara; Ketchum, Karen; Kinsinger, Christopher; Kuhn, Eric; Liebler, Daniel; Liu, Tao; Loss, Michael; MacCoss, Michael; Qian, Weijun; Rivers, Robert; Rodland, Karin D.; Ruggles, Kelly; Scott, Mitchell; Smith, Richard D.; Thomas, Stefani N.; Townsend, Reid; Whiteley, Gordon; Wu, Chaochao; Zhang, Hui; Zhang, Zhen; Rodriguez, Henry; Paulovich, Amanda G.

    2016-02-12

    The Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the National Cancer Institute (NCI) has launched an Assay Portal (http://assays.cancer.gov) to serve as an open-source repository of well-characterized targeted proteomic assays. The portal is designed to curate and disseminate highly characterized, targeted mass spectrometry (MS)-based assays by providing detailed assay performance characterization data, standard operating procedures, and access to reagents. Assay content is accessed via the portal through queries to find assays targeting proteins associated with specific cellular pathways, protein complexes, or specific chromosomal regions. The position of the peptide analytes for which there are available assays are mapped relative to other features of interest in the protein, such as sequence domains, isoforms, single nucleotide polymorphisms, and post-translational modifications. The overarching goals are to enable robust quantification of all human proteins and to standardize the quantification of targeted MS-based assays to ultimately enable harmonization of results over time and across laboratories.

  3. Genomic profiling of murine mammary tumors identifies potential personalized drug targets for p53-deficient mammary cancers

    Directory of Open Access Journals (Sweden)

    Adam D. Pfefferle

    2016-07-01

    Full Text Available Targeted therapies against basal-like breast tumors, which are typically ‘triple-negative breast cancers (TNBCs’, remain an important unmet clinical need. Somatic TP53 mutations are the most common genetic event in basal-like breast tumors and TNBC. To identify additional drivers and possible drug targets of this subtype, a comparative study between human and murine tumors was performed by utilizing a murine Trp53-null mammary transplant tumor model. We show that two subsets of murine Trp53-null mammary transplant tumors resemble aspects of the human basal-like subtype. DNA-microarray, whole-genome and exome-based sequencing approaches were used to interrogate the secondary genetic aberrations of these tumors, which were then compared to human basal-like tumors to identify conserved somatic genetic features. DNA copy-number variation produced the largest number of conserved candidate personalized drug targets. These candidates were filtered using a DNA-RNA Pearson correlation cut-off and a requirement that the gene was deemed essential in at least 5% of human breast cancer cell lines from an RNA-mediated interference screen database. Five potential personalized drug target genes, which were spontaneously amplified loci in both murine and human basal-like tumors, were identified: Cul4a, Lamp1, Met, Pnpla6 and Tubgcp3. As a proof of concept, inhibition of Met using crizotinib caused Met-amplified murine tumors to initially undergo complete regression. This study identifies Met as a promising drug target in a subset of murine Trp53-null tumors, thus identifying a potential shared driver with a subset of human basal-like breast cancers. Our results also highlight the importance of comparative genomic studies for discovering personalized drug targets and for providing a preclinical model for further investigations of key tumor signaling pathways.

  4. CRISPRseek: a bioconductor package to identify target-specific guide RNAs for CRISPR-Cas9 genome-editing systems.

    Directory of Open Access Journals (Sweden)

    Lihua J Zhu

    Full Text Available CRISPR-Cas systems are a diverse family of RNA-protein complexes in bacteria that target foreign DNA sequences for cleavage. Derivatives of these complexes have been engineered to cleave specific target sequences depending on the sequence of a CRISPR-derived guide RNA (gRNA and the source of the Cas9 protein. Important considerations for the design of gRNAs are to maximize aimed activity at the desired target site while minimizing off-target cleavage. Because of the rapid advances in the understanding of existing CRISPR-Cas9-derived RNA-guided nucleases and the development of novel RNA-guided nuclease systems, it is critical to have computational tools that can accommodate a wide range of different parameters for the design of target-specific RNA-guided nuclease systems. We have developed CRISPRseek, a highly flexible, open source software package to identify gRNAs that target a given input sequence while minimizing off-target cleavage at other sites within any selected genome. CRISPRseek will identify potential gRNAs that target a sequence of interest for CRISPR-Cas9 systems from different bacterial species and generate a cleavage score for potential off-target sequences utilizing published or user-supplied weight matrices with position-specific mismatch penalty scores. Identified gRNAs may be further filtered to only include those that occur in paired orientations for increased specificity and/or those that overlap restriction enzyme sites. For applications where gRNAs are desired to discriminate between two related sequences, CRISPRseek can rank gRNAs based on the difference between predicted cleavage scores in each input sequence. CRISPRseek is implemented as a Bioconductor package within the R statistical programming environment, allowing it to be incorporated into computational pipelines to automate the design of gRNAs for target sequences identified in a wide variety of genome-wide analyses. CRISPRseek is available under the GNU General

  5. A Proteomic Approach to Identify Phosphorylation-Dependent Targets of BRCT Domains

    Science.gov (United States)

    2008-03-01

    targets To develop the BiFC technology for screening for BRCT- BRCA1 interacting proteins in mammalian cells, we constructed expression...domain To screen for BRCA1-BRCT interacting proteins using BiFC, we undertook several approaches. In the last report, we described a reduction approach...candidate BRCT- interacting proteins . Indeed, we obtained many cell clones that exhibited either strong or weak fluorescence, indicating that these

  6. Multiplatform molecular profiling identifies potentially targetable biomarkers in malignant phyllodes tumors of the breast

    OpenAIRE

    Gatalica, Zoran; Vranic, Semir; Ghazalpour, Anatole; Xiu, Joanne; Ocal, Idris Tolgay; McGill, John; Bender, Ryan P.; Discianno, Erin; Schlum, Aaron; Sanati, Souzan; Palazzo, Juan; Reddy, Sandeep; Pockaj, Barbara

    2015-01-01

    Malignant phyllodes tumor is a rare breast malignancy with sarcomatous overgrowth and with limited effective treatment options for recurrent and metastatic cases. Recent clinical trials indicated a potential for anti-angiogenic, anti-EGFR and immunotherapeutic approaches for patients with sarcomas, which led us to investigate these and other targetable pathways in malignant phyllodes tumor of the breast. Thirty-six malignant phyllodes tumors (including 8 metastatic tumors with two cases havin...

  7. Quantitative liver proteomics identifies FGF19 targets that couple metabolism and proliferation

    OpenAIRE

    Massafra, Vittoria; Milona, Alexandra; VOS, HARMJAN R.; Burgering, Boudewijn M. T.; van Mil, Saskia W. C.

    2017-01-01

    Fibroblast growth factor 19 (FGF19) is a gut-derived peptide hormone that is produced following activation of Farnesoid X Receptor (FXR). FGF19 is secreted and signals to the liver, where it contributes to the homeostasis of bile acid (BA), lipid and carbohydrate metabolism. FGF19 is a promising therapeutic target for the metabolic syndrome and cholestatic diseases, but enthusiasm for its use has been tempered by FGF19-mediated induction of proliferation and hepatocellular carcinoma. To infor...

  8. Disruption of Esrom and Ryk identifies the roof plate boundary as an intermediate target for commissure formation.

    Science.gov (United States)

    Hendricks, Michael; Mathuru, Ajay Sriram; Wang, Hui; Silander, Olin; Kee, Michelle Zhi Ling; Jesuthasan, Suresh

    2008-02-01

    Growth cones are guided to their final destination by intermediate targets. Here, we identify intermediate targets and signaling components acting on zebrafish habenula commissural axons. Live imaging establishes that axons pause at the medial habenula before and after crossing the roof plate. esrom mutants axons fail to advance beyond the ipsilateral medial habenula. Tsc2 function is reduced in mutant axons, indicating cell autonomous defects in signaling. Consistent with signaling properties changing outside the roof plate, EphB is surface localized on axon segments within a zone demarcated by the medial habenula. wnt4a is expressed in the medial habenula and morpholino knockdown causes loss of the commissure. Electroporation of truncated Ryk causes axons to reenter the midline after reaching the contralateral habenula. These data identify Esrom as a mediator of growth cone navigation at an intermediate target and underscore the importance of midline boundaries as signaling centers for commissure formation.

  9. Spiroindolines Identify the Vesicular Acetylcholine Transporter as a Novel Target for Insecticide Action

    Science.gov (United States)

    Sluder, Ann; Shah, Sheetal; Cassayre, Jérôme; Clover, Ralph; Maienfisch, Peter; Molleyres, Louis-Pierre; Hirst, Elizabeth A.; Flemming, Anthony J.; Shi, Min; Cutler, Penny; Stanger, Carole; Roberts, Richard S.; Hughes, David J.; Flury, Thomas; Robinson, Michael P.; Hillesheim, Elke; Pitterna, Thomas; Cederbaum, Fredrik; Worthington, Paul A.; Crossthwaite, Andrew J.; Windass, John D.; Currie, Richard A.; Earley, Fergus G. P.

    2012-01-01

    The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines) encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family. PMID:22563457

  10. Spiroindolines identify the vesicular acetylcholine transporter as a novel target for insecticide action.

    Directory of Open Access Journals (Sweden)

    Ann Sluder

    Full Text Available The efficacy of all major insecticide classes continues to be eroded by the development of resistance mediated, in part, by selection of alleles encoding insecticide insensitive target proteins. The discovery of new insecticide classes acting at novel protein binding sites is therefore important for the continued protection of the food supply from insect predators, and of human and animal health from insect borne disease. Here we describe a novel class of insecticides (Spiroindolines encompassing molecules that combine excellent activity against major agricultural pest species with low mammalian toxicity. We confidently assign the vesicular acetylcholine transporter as the molecular target of Spiroindolines through the combination of molecular genetics in model organisms with a pharmacological approach in insect tissues. The vesicular acetylcholine transporter can now be added to the list of validated insecticide targets in the acetylcholine signalling pathway and we anticipate that this will lead to the discovery of novel molecules useful in sustaining agriculture. In addition to their potential as insecticides and nematocides, Spiroindolines represent the only other class of chemical ligands for the vesicular acetylcholine transporter since those based on the discovery of vesamicol over 40 years ago, and as such, have potential to provide more selective tools for PET imaging in the diagnosis of neurodegenerative disease. They also provide novel biochemical tools for studies of the function of this protein family.

  11. Chemical genetic strategy identifies histone deacetylase 1 (HDAC1) and HDAC2 as therapeutic targets in sickle cell disease

    National Research Council Canada - National Science Library

    James E. Bradner; Raymond Mak; Shyam K. Tanguturi; Ralph Mazitschek; Stephen J. Haggarty; Kenneth Ross; Cindy Y. Chang; Jocelyn Bosco; Nathan West; Elizabeth Morse; Katherine Lin; John Paul Shen; Nicholas P. Kwiatkowski; Nele Gheldof; Job Dekker; Daniel J. DeAngelo; Steven A. Carr; Stuart L. Schreiber; Todd R. Golub; Benjamin L. Ebert

    2010-01-01

    .... Using a chemical genetic strategy combining focused libraries of biased chemical probes and reverse genetics by RNA interference, we have identified HDAC1 and HDAC2 as molecular targets mediating fetal hemoglobin induction. Our findings suggest the potential of isoform-selective inhibitors of HDAC1 and HDAC2 for the treatment of sickle cell disease.

  12. Mutations in the DDR2 Kinase Gene identify a Novel therapeutic target in squamous cell lung cancer

    NARCIS (Netherlands)

    Hammerman, Peter S.; Sos, Martin L.; Ramos, Alex H.; Xu, Chunxiao; Dutt, Amit; Zhou, Wenjun; Brace, Lear E.; Woods, Brittany A.; Lin, Wenchu; Zhang, Jianming; Deng, Xianming; Lim, Sang Min; Heynck, Stefanie; Peifer, Martin; Simard, Jeffrey R.; Lawrence, Michael S.; Onofrio, Robert C.; Salvesen, Helga B.; Seidel, Danila; Zander, Thomas; Heuckmann, Johannes M.; Soltermann, Alex; Moch, Holger; Koker, Mirjam; Leenders, Frauke; Gabler, Franziska; Querings, Silvia; Ansen, Sascha; Brambilla, Elisabeth; Brambilla, Christian; Lorimier, Philippe; Brustugun, Odd Terje; Helland, Aslaug; Petersen, Iver; Clement, Joachim H.; Groen, Harry; Timens, Wim; Sietsma, Hannie; Stoelben, Erich; Wolf, Juergen; Beer, David G.; Tsao, Ming Sound; Hanna, Megan; Hatton, Charles; Eck, Michael J.; Janne, Pasi A.; Johnson, Bruce E.; Winckler, Wendy; Greulich, Heidi; Bass, Adam J.; Cho, Jeonghee; Rauh, Daniel; Gray, Nathanael S.; Wong, Kwok-Kin; Haura, Eric B.; Thomas, Roman K.; Meyerson, Matthew

    2011-01-01

    Although genomically targeted therapies have improved outcomes for patients with lung adenocarcinoma, little is known about the genomic alterations that drive squamous cell cancer (SCC) of the lung. Sanger sequencing of the tyrosine kinome identified mutations in the DDR2 kinase gene in 3.8% of lung

  13. Methods to identify the target population : implications for prescribing quality indicators

    NARCIS (Netherlands)

    Martirosyan, Liana; Arah, Onyebuchi A.; Haaijer-Ruskamp, Flora M.; Braspenning, Joze; Denig, Petra

    2010-01-01

    Background: Information on prescribing quality is increasingly used by policy makers, insurance companies and health care providers. For reliable assessment of prescribing quality it is important to correctly identify the patients eligible for recommended treatment. Often either diagnostic codes or

  14. An integrated structure- and system-based framework to identify new targets of metabolites and known drugs

    KAUST Repository

    Naveed, Hammad

    2015-08-18

    Motivation: The inherent promiscuity of small molecules towards protein targets impedes our understanding of healthy versus diseased metabolism. This promiscuity also poses a challenge for the pharmaceutical industry as identifying all protein targets is important to assess (side) effects and repositioning opportunities for a drug. Results: Here, we present a novel integrated structure- and system-based approach of drug-target prediction (iDTP) to enable the large-scale discovery of new targets for small molecules, such as pharmaceutical drugs, co-factors and metabolites (collectively called ‘drugs’). For a given drug, our method uses sequence order–independent structure alignment, hierarchical clustering, and probabilistic sequence similarity to construct a probabilistic pocket ensemble (PPE) that captures promiscuous structural features of different binding sites on known targets. A drug’s PPE is combined with an approximation of its delivery profile to reduce false positives. In our cross-validation study, we use iDTP to predict the known targets of eleven drugs, with 63% sensitivity and 81% specificity. We then predicted novel targets for these drugs—two that are of high pharmacological interest, the nuclear receptor PPARγ and the oncogene Bcl-2, were successfully validated through in vitro binding experiments. Our method is broadly applicable for the prediction of protein-small molecule interactions with several novel applications to biological research and drug development.

  15. Soft computing model for optimized siRNA design by identifying off target possibilities using artificial neural network model.

    Science.gov (United States)

    Murali, Reena; John, Philips George; Peter S, David

    2015-05-15

    The ability of small interfering RNA (siRNA) to do posttranscriptional gene regulation by knocking down targeted genes is an important research topic in functional genomics, biomedical research and in cancer therapeutics. Many tools had been developed to design exogenous siRNA with high experimental inhibition. Even though considerable amount of work has been done in designing exogenous siRNA, design of effective siRNA sequences is still a challenging work because the target mRNAs must be selected such that their corresponding siRNAs are likely to be efficient against that target and unlikely to accidentally silence other transcripts due to sequence similarity. In some cases, siRNAs may tolerate mismatches with the target mRNA, but knockdown of genes other than the intended target could make serious consequences. Hence to design siRNAs, two important concepts must be considered: the ability in knocking down target genes and the off target possibility on any nontarget genes. So before doing gene silencing by siRNAs, it is essential to analyze their off target effects in addition to their inhibition efficacy against a particular target. Only a few methods have been developed by considering both efficacy and off target possibility of siRNA against a gene. In this paper we present a new design of neural network model with whole stacking energy (ΔG) that enables to identify the efficacy and off target effect of siRNAs against target genes. The tool lists all siRNAs against a particular target with their inhibition efficacy and number of matches or sequence similarity with other genes in the database. We could achieve an excellent performance of Pearson Correlation Coefficient (R=0. 74) and Area Under Curve (AUC=0.906) when the threshold of whole stacking energy is ≥-34.6 kcal/mol. To the best of the author's knowledge, this is one of the best score while considering the "combined efficacy and off target possibility" of siRNA for silencing a gene. The proposed model

  16. Identified central neurons convey a mitogenic signal from a peripheral target to the CNS.

    Science.gov (United States)

    Becker, T S; Bothe, G; Berliner, A J; Macagno, E R

    1996-08-01

    Regulation of central neurogenesis by a peripheral target has been previously demonstrated in the ventral nerve cord of the leech Hirudo medicinalis (Baptista, C. A., Gershon, T. R. and Macagno, E. R. (1990). Nature 346, 855-858) Specifically, innervation of the male genitalia by the fifth and sixth segmental ganglia (the sex ganglia) was shown to trigger the birth of several hundred central neurons (PIC neurons) in these ganglia. As reported here, removal of the target early during induction shows that PIC neurons can be independently induced in each side of a ganglion, indicating that the inductive signal is both highly localized and conveyed to each hemiganglion independently. Further, since recent observations (Becker, T., Berliner, A. J., Nitabach, M. N., Gan, W.-B. and Macagno, E. R. (1995). Development, 121, 359-369) had indicated that efferent projections are probably involved in this phenomenon, we individually ablated all possible candidates, which led to the identification of two central neurons that appear to play significant roles in conveying the inductive signal to the CNS. Ablation of a single ML neuron reduced cell proliferation in its own hemiganglion by nearly 50%, on the average. In contrast, proliferation on the opposite side of the ganglion increased by about 25%, suggesting the possibility of a compensatory response by the remaining contralateral ML neuron. Simultaneous ablation of both ML neurons in a sex ganglion caused similar reductions in cell proliferation in each hemiganglion. Deletion of a single AL neuron produced a weaker (7%) but nonetheless reproducible reduction. Ablation of the other nine central neurons that might have been involved in PIC neuron induction had no detectable effect. Both ML and AL neurons exhibit ipsilateral peripheral projections, and both arborize mostly in the hemiganglion where they reside. Thus, we conclude that peripheral regulation of central neurogenesis is mediated in the leech by inductive signals

  17. Identifying High Potential Well Targets with 3D Seismic and Mineralogy

    Energy Technology Data Exchange (ETDEWEB)

    Mellors, R. J. [Lawrence Livermore National Lab. (LLNL), Livermore, CA (United States)

    2015-10-30

    Seismic reflection the primary tool used in petroleum exploration and production, but use in geothermal exploration is less standard, in part due to cost but also due to the challenges in identifying the highly-permeable zones essential for economic hydrothermal systems [e.g. Louie et al., 2011; Majer, 2003]. Newer technology, such as wireless sensors and low-cost high performance computing, has helped reduce the cost and effort needed to conduct 3D surveys. The second difficulty, identifying permeable zones, has been less tractable so far. Here we report on the use of seismic attributes from a 3D seismic survey to identify and map permeable zones in a hydrothermal area.

  18. Methods for Identifying Ligands that Target Nucleic Acid Molecules and Nucleic Acid Structural Motifs

    Science.gov (United States)

    Disney, Matthew D. (Inventor); Childs-Disney, Jessica L. (Inventor)

    2017-01-01

    Disclosed are methods for identifying a nucleic acid (e.g., RNA, DNA, etc.) motif which interacts with a ligand. The method includes providing a plurality of ligands immobilized on a support, wherein each particular ligand is immobilized at a discrete location on the support; contacting the plurality of immobilized ligands with a nucleic acid motif library under conditions effective for one or more members of the nucleic acid motif library to bind with the immobilized ligands; and identifying members of the nucleic acid motif library that are bound to a particular immobilized ligand. Also disclosed are methods for selecting, from a plurality of candidate ligands, one or more ligands that have increased likelihood of binding to a nucleic acid molecule comprising a particular nucleic acid motif, as well as methods for identifying a nucleic acid which interacts with a ligand.

  19. Chemical screen identifies FDA-approved drugs and target pathways that induce precocious pancreatic endocrine differentiation.

    Science.gov (United States)

    Rovira, Meritxell; Huang, Wei; Yusuff, Shamila; Shim, Joong Sup; Ferrante, Anthony A; Liu, Jun O; Parsons, Michael J

    2011-11-29

    Pancreatic β-cells are an essential source of insulin and their destruction because of autoimmunity causes type I diabetes. We conducted a chemical screen to identify compounds that would induce the differentiation of insulin-producing β-cells in vivo. To do this screen, we brought together the use of transgenic zebrafish as a model of β-cell differentiation, a unique multiwell plate that allows easy visualization of lateral views of swimming larval fish and a library of clinical drugs. We identified six hits that can induce precocious differentiation of secondary islets in larval zebrafish. Three of these six hits were known drugs with a considerable background of published data on mechanism of action. Using pharmacological approaches, we have identified and characterized two unique pathways in β-cell differentiation in the zebrafish, including down-regulation of GTP production and retinoic acid biosynthesis.

  20. Exploring the surfaceome of Ewing sarcoma identifies a new and unique therapeutic target

    Science.gov (United States)

    Town, Jennifer; Pais, Helio; Harrison, Sally; Bataille, Carole; Bunjobpol, Wilawan; Zhang, Jing; Rabbitts, Terence H.

    2016-01-01

    The cell surface proteome of tumors mediates the interface between the transformed cells and the general microenvironment, including interactions with stromal cells in the tumor niche and immune cells such as T cells. In addition, the cell surface proteome of individual cancers defines biomarkers for that tumor type and potential proteins that can be the target of antibody-mediated therapy. We have used next-generation deep RNA sequencing (RNA-seq) coupled to an in-house database of genes encoding cell surface proteins (herein referred to as the surfaceome) as a tool to define a cell surface proteome of Ewing sarcoma compared with progenitor mesenchymal stem cells. This subtractive RNA-seq analysis revealed a specific surfaceome of Ewing and showed unexpectedly that the leucine-rich repeat and Ig domain protein 1 (LINGO1) is expressed in over 90% of Ewing sarcoma tumors, but not expressed in any other somatic tissue apart from the brain. We found that the LINGO1 protein acts as a gateway protein internalizing into the tumor cells when engaged by antibody and can carry antibody conjugated with drugs to kill Ewing sarcoma cells. Therefore, LINGO1 is a new, unique, and specific biomarker and drug target for the treatment of Ewing sarcoma. PMID:26979953

  1. MEDICI: Mining Essentiality Data to Identify Critical Interactions for Cancer Drug Target Discovery and Development.

    Science.gov (United States)

    Harati, Sahar; Cooper, Lee A D; Moran, Josue D; Giuste, Felipe O; Du, Yuhong; Ivanov, Andrei A; Johns, Margaret A; Khuri, Fadlo R; Fu, Haian; Moreno, Carlos S

    2017-01-01

    Protein-protein interactions (PPIs) mediate the transmission and regulation of oncogenic signals that are essential to cellular proliferation and survival, and thus represent potential targets for anti-cancer therapeutic discovery. Despite their significance, there is no method to experimentally disrupt and interrogate the essentiality of individual endogenous PPIs. The ability to computationally predict or infer PPI essentiality would help prioritize PPIs for drug discovery and help advance understanding of cancer biology. Here we introduce a computational method (MEDICI) to predict PPI essentiality by combining gene knockdown studies with network models of protein interaction pathways in an analytic framework. Our method uses network topology to model how gene silencing can disrupt PPIs, relating the unknown essentialities of individual PPIs to experimentally observed protein essentialities. This model is then deconvolved to recover the unknown essentialities of individual PPIs. We demonstrate the validity of our approach via prediction of sensitivities to compounds based on PPI essentiality and differences in essentiality based on genetic mutations. We further show that lung cancer patients have improved overall survival when specific PPIs are no longer present, suggesting that these PPIs may be potentially new targets for therapeutic development. Software is freely available at https://github.com/cooperlab/MEDICI. Datasets are available at https://ctd2.nci.nih.gov/dataPortal.

  2. Analysis of Sequenced Genomes of Xanthomonas perforans Identifies Candidate Targets for Resistance Breeding in Tomato.

    Science.gov (United States)

    Timilsina, Sujan; Abrahamian, Peter; Potnis, Neha; Minsavage, Gerald V; White, Frank F; Staskawicz, Brian J; Jones, Jeffrey B; Vallad, Gary E; Goss, Erica M

    2016-10-01

    Bacterial disease management is a challenge for modern agriculture due to rapid changes in pathogen populations. Genome sequences for hosts and pathogens provide detailed information that facilitates effector-based breeding strategies. Tomato genotypes have gene-for-gene resistance to the bacterial spot pathogen Xanthomonas perforans. The bacterial spot populations in Florida shifted from tomato race 3 to 4, such that the corresponding tomato resistance gene no longer recognizes the effector protein AvrXv3. Genome sequencing showed variation in effector profiles among race 4 strains collected in 2006 and 2012 and compared with a race 3 strain collected in 1991. We examined variation in putative targets of resistance among Florida strains of X. perforans collected from 1991 to 2006. Consistent with race change, avrXv3 was present in race 3 strains but nonfunctional in race 4 strains due to multiple independent mutations. Effectors xopJ4 and avrBs2 were unchanged in all strains. The effector avrBsT was absent in race 3 strains collected in the 1990s but present in race 3 strains collected in 2006 and nearly all race 4 strains. These changes in effector profiles suggest that xopJ4 and avrBsT are currently the best targets for resistance breeding against bacterial spot in tomato.

  3. Identifying lipidic emulsomes for improved oxcarbazepine brain targeting: In vitro and rat in vivo studies.

    Science.gov (United States)

    El-Zaafarany, Ghada M; Soliman, Mahmoud E; Mansour, Samar; Awad, Gehanne A S

    2016-04-30

    Lipid-based nanovectors offer effective carriers for brain delivery by improving drug potency and reducing off-target effects. Emulsomes are nano-triglyceride (TG) carriers formed of lipid cores supported by at least one phospholipid (PC) sheath. Due to their surface active properties, PC forms bilayers at the aqueous interface, thereby enabling encapsulated drug to benefit from better bioavailability and stability. Emulsomes of oxcarbazepine (OX) were prepared, aimed to offer nanocarriers for nasal delivery for brain targeting. Different TG cores (Compritol(®), tripalmitin, tristearin and triolein) and soya phosphatidylcholine in different amounts and ratios were used for emulsomal preparation. Particles were modulated to generate nanocarriers with suitable size, charge, encapsulation efficiency and prolonged release. Cytotoxicity and pharmacokinetic studies were also implemented. Nano-spherical OX-emulsomes with maximal encapsulation of 96.75% were generated. Stability studies showed changes within 30.6% and 11.2% in the size and EE% after 3 months. MTT assay proved a decrease in drug toxicity by its encapsulation in emulsomes. Incorporation of OX into emulsomes resulted in stable nanoformulations. Tailoring emulsomes properties by modulating the surface charge and particle size produced a stable system for the lipophilic drug with a prolonged release profile and mean residence time and proved direct nose-to-brain transport in rats.

  4. Targeted screening programmes in COPD: how to identify individuals with α1-antitrypsin deficiency.

    Science.gov (United States)

    Chorostowska-Wynimko, Joanna

    2015-03-01

    α1-antitrypsin deficiency (AATD) is a significantly under-recognised autosomal genetic disorder with individuals being clinically diagnosed. Moreover, rigorous genetic epidemiological data regarding AATD are lacking. The majority of findings come from the USA and Western Europe, and no information is available for many countries. To address this concern, an α1-antitrypsin (AAT) laboratory was set up in 2009 at the National Institute of Tuberculosis and Lung Diseases (Warsaw, Poland). In 2010, an AATD screening programme targeting patients with respiratory disorders was initiated in Poland. This targeted survey has provided valuable information regarding AAT-deficient genotypes, clinical disease and levels of expertise at the physician level. After 4 years, almost 2500 patients with chronic obstructive pulmonary disorders have been screened and, in this cohort, ∼13% had AATD alleles. In these patients, the detection frequency for S and Z alleles was four times greater, and the frequency of homozygous PI*ZZ was 16 times greater than that of the general population. These results highlight the need to build awareness in the medical community, and the project is currently being extended to cover central Eastern Europe, with the creation of the Central Eastern European Alpha-1 Antitrypsin Network.

  5. Identifying and targeting cancer stem cells in the treatment of gastric cancer.

    Science.gov (United States)

    Bekaii-Saab, Tanios; El-Rayes, Bassel

    2017-04-15

    Current treatment regimens for gastric cancer are not adequate. Cancer stem cells (CSCs) may be a key driving factor for growth and metastasis of this tumor type. In contrast to the conventional clonal evolution hypothesis, CSCs can initiate tumor formation, self-renew, and differentiate into tumor-propagating cells. Because gastric cancer can originate from CSCs, it is necessary to review current targets of signaling pathways for CSCs in gastric cancer that are being studied in clinical trials. These pathways are known to regulate the self-renewal and differentiation process in gastric CSCs. A better understanding of the clinical results of trials that target gastric CSCs will lead to better outcomes for patients with gastric cancer. Cancer 2017;123:1303-1312. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society. This is an open access article under the terms of the Creative Commons Attribution NonCommercial License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. © 2017 The Authors. Cancer published by Wiley Periodicals, Inc. on behalf of American Cancer Society.

  6. Characterization of plant nematode genes: identifying targets for a transgenic defence.

    Science.gov (United States)

    Lilley, C J; Urwin, P E; Atkinson, H J

    1999-01-01

    Current control of plant parasitic nematodes often relies on highly toxic and environmentally harmful nematicides. As their use becomes increasingly restricted there is an urgent need to develop crop varieties with resistance to nematodes. The limitations surrounding conventional plant breeding ensure there is a clear opportunity for transgenic resistance to lessen current dependence on chemical control. The increasing use of molecular biology techniques in the field of plant nematology is now providing useful information for the design of novel defences to meet the new needs. Plant responses to parasitism are being investigated at the molecular level and nematode gene products that could be targets for a direct anti-nematode defence are being characterized. The potential of an anti-feedant approach to nematode control has been demonstrated. It is based on the transgenic expression of proteinase inhibitors. The rational development of this strategy involves characterization of nematode proteinase genes and optimization of inhibitors by protein engineering. Durability of the resistance can be enhanced by stacking transgenes directed at different nematode targets.

  7. New mutations in chronic lymphocytic leukemia identified by target enrichment and deep sequencing.

    Directory of Open Access Journals (Sweden)

    Elena Doménech

    Full Text Available Chronic lymphocytic leukemia (CLL is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR, and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3 have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.

  8. New mutations in chronic lymphocytic leukemia identified by target enrichment and deep sequencing.

    Science.gov (United States)

    Doménech, Elena; Gómez-López, Gonzalo; Gzlez-Peña, Daniel; López, Mar; Herreros, Beatriz; Menezes, Juliane; Gómez-Lozano, Natalia; Carro, Angel; Graña, Osvaldo; Pisano, David G; Domínguez, Orlando; García-Marco, José A; Piris, Miguel A; Sánchez-Beato, Margarita

    2012-01-01

    Chronic lymphocytic leukemia (CLL) is a heterogeneous disease without a well-defined genetic alteration responsible for the onset of the disease. Several lines of evidence coincide in identifying stimulatory and growth signals delivered by B-cell receptor (BCR), and co-receptors together with NFkB pathway, as being the driving force in B-cell survival in CLL. However, the molecular mechanism responsible for this activation has not been identified. Based on the hypothesis that BCR activation may depend on somatic mutations of the BCR and related pathways we have performed a complete mutational screening of 301 selected genes associated with BCR signaling and related pathways using massive parallel sequencing technology in 10 CLL cases. Four mutated genes in coding regions (KRAS, SMARCA2, NFKBIE and PRKD3) have been confirmed by capillary sequencing. In conclusion, this study identifies new genes mutated in CLL, all of them in cases with progressive disease, and demonstrates that next-generation sequencing technologies applied to selected genes or pathways of interest are powerful tools for identifying novel mutational changes.

  9. A Comprehensive Approach to Identifying Intervention Targets for Patient-Safety Improvement in a Hospital Setting

    Science.gov (United States)

    Cunningham, Thomas R.; Geller, E. Scott

    2012-01-01

    Despite differences in approaches to organizational problem solving, healthcare managers and organizational behavior management (OBM) practitioners share a number of practices, and connecting healthcare management with OBM may lead to improvements in patient safety. A broad needs-assessment methodology was applied to identify patient-safety…

  10. Proteomic Analysis of Kveim Reagent Identifies Targets of Cellular Immunity in Sarcoidosis.

    Science.gov (United States)

    Eberhardt, Christian; Thillai, Muhunthan; Parker, Robert; Siddiqui, Nazneen; Potiphar, Lee; Goldin, Rob; Timms, John F; Wells, Athol U; Kon, Onn M; Wickremasinghe, Melissa; Mitchell, Donald; Weeks, Mark E; Lalvani, Ajit

    2017-01-01

    Kveim-reagent (Kv) skin testing was a historical method of diagnosing sarcoidosis. Intradermal injection of treated sarcoidosis spleen tissue resulted in a granuloma response at injection site by 4-6 weeks. Previous work indicates proteins as the possible trigger of this reaction. We aimed to identify Kv-specific proteins and characterise the ex vivo response of Peripheral Blood Mononuclear Cells (PBMCs) from sarcoidosis, tuberculosis and healthy control patients when stimulated with both Kv and selected Kv-specific proteins. Kv extracts were separated by 1D-SDS-PAGE and 2D-DIGE and then underwent mass spectrometric analysis for protein identification. Sarcoidosis and control PBMCs were first stimulated with Kv and then with three selected recombinant protein candidates which were identified from the proteomic analysis. PBMC secreted cytokines were subsequently measured by Multiplex Cytokine Assay. We observed significantly increased IFN-γ and TNF-α secretion from Kv-stimulated PBMCs of sarcoidosis patients vs. PBMCs from healthy volunteers (IFN-γ: 207.2 pg/mL vs. 3.86 pg/mL, p = 0.0018; TNF-α: 2375 pg/mL vs. 42.82 pg/mL, p = 0.0003). Through proteomic approaches we then identified 74 sarcoidosis tissue-specific proteins. Of these, 3 proteins (vimentin, tubulin and alpha-actinin-4) were identified using both 1D-SDS-PAGE and 2D-DIGE. Data are available via ProteomeXchange with identifier PXD005150. Increased cytokine secretion was subsequently observed with vimentin stimulation of sarcoidosis PBMCs vs. tuberculosis PBMCs (IFN-γ: 396.6 pg/mL vs 0.1 pg/mL, p = 0.0009; TNF-α: 1139 pg/mL vs 0.1 pg/mL, psarcoidosis PBMCs compared to PBMCs from healthy controls (IFN-γ: 396.6 pg/mL vs. 0.1 pg/mL, p = 0.014; TNF-α: 1139 pg/mL vs 42.29 pg/mL, p = 0.027). No difference was found in cytokine secretion between sarcoidosis and control PBMCs when stimulated with either tubulin or alpha-actinin-4. Stimulation with both Kveim reagent and vimentin induces a specific pro

  11. Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens

    Directory of Open Access Journals (Sweden)

    Prabodhika Mallikaratchy

    2017-01-01

    Full Text Available The demand has increased for sophisticated molecular tools with improved detection limits. Such molecules should be simple in structure, yet stable enough for clinical applications. Nucleic acid aptamers (NAAs represent a class of molecules able to meet this demand. In particular, aptamers, a class of small nucleic acid ligands that are composed of single-stranded modified/unmodified RNA/DNA molecules, can be evolved from a complex library using Systematic Evolution of Ligands by EXponential enrichment (SELEX against almost any molecule. Since its introduction in 1990, in stages, SELEX technology has itself undergone several modifications, improving selection and broadening the repertoire of targets. This review summarizes these milestones that have pushed the field forward, allowing researchers to generate aptamers that can potentially be applied as therapeutic and diagnostic agents.

  12. Human visual search does not maximize the post-saccadic probability of identifying targets.

    Directory of Open Access Journals (Sweden)

    Camille Morvan

    2012-02-01

    Full Text Available Researchers have conjectured that eye movements during visual search are selected to minimize the number of saccades. The optimal Bayesian eye movement strategy minimizing saccades does not simply direct the eye to whichever location is judged most likely to contain the target but makes use of the entire retina as an information gathering device during each fixation. Here we show that human observers do not minimize the expected number of saccades in planning saccades in a simple visual search task composed of three tokens. In this task, the optimal eye movement strategy varied, depending on the spacing between tokens (in the first experiment or the size of tokens (in the second experiment, and changed abruptly once the separation or size surpassed a critical value. None of our observers changed strategy as a function of separation or size. Human performance fell far short of ideal, both qualitatively and quantitatively.

  13. Evolution of Complex Target SELEX to Identify Aptamers against Mammalian Cell-Surface Antigens.

    Science.gov (United States)

    Mallikaratchy, Prabodhika

    2017-01-30

    The demand has increased for sophisticated molecular tools with improved detection limits. Such molecules should be simple in structure, yet stable enough for clinical applications. Nucleic acid aptamers (NAAs) represent a class of molecules able to meet this demand. In particular, aptamers, a class of small nucleic acid ligands that are composed of single-stranded modified/unmodified RNA/DNA molecules, can be evolved from a complex library using Systematic Evolution of Ligands by EXponential enrichment (SELEX) against almost any molecule. Since its introduction in 1990, in stages, SELEX technology has itself undergone several modifications, improving selection and broadening the repertoire of targets. This review summarizes these milestones that have pushed the field forward, allowing researchers to generate aptamers that can potentially be applied as therapeutic and diagnostic agents.

  14. The Use of the Target Cancellation Task to Identify Eloquent Visuospatial Regions in Awake Craniotomies: Technical Note.

    Science.gov (United States)

    Conner, Andrew K; Glenn, Chad; Burks, Joshua D; McCoy, Tressie; Bonney, Phillip A; Chema, Ahmed A; Case, Justin L; Brunner, Scott; Baker, Cordell; Sughrue, Michael

    2016-11-17

    The success of awake craniotomies relies on the patient's performance of function-specific tasks that are simple, quick, and reproducible. Intraoperative identification of visuospatial function through cortical and subcortical mapping has utilized a variety of intraoperative tests, each with its own benefits and drawbacks. In light of this, we developed a simple software program that aids in preventing neglect by simulating a target-cancellation task on a portable electronic device. In this report, we describe the interactive target cancellation task and have reviewed seven consecutive patients who underwent awake craniotomy for parietal and/or posterior temporal infiltrating brain tumors of the non-dominant hemisphere. Each of these patients performed target cancellation and line bisection tasks intraoperatively. The outcomes of each patient and testing scenario are described. Positive intraoperative cortical and subcortical sites involved with visuospatial processing were identified in three of the seven patients using the target cancellation and confirmed utilizing the line-bisection task. No identification of visuospatial function was accomplished utilizing the line-bisection task alone. Complete visuospatial function mapping was completed in less than 10 minutes in all patients. No patients had preoperative or postoperative hemineglect. Our findings highlight the feasibility of the target cancellation technique for use during awake craniotomy to aid in avoiding postoperative hemineglect. Target cancellation may offer an alternative method of cortical and subcortical visuospatial mapping in patients unable to perform other commonly used modalities.

  15. A computational study of the Warburg effect identifies metabolic targets inhibiting cancer migration.

    Science.gov (United States)

    Yizhak, Keren; Le Dévédec, Sylvia E; Rogkoti, Vasiliki Maria; Baenke, Franziska; de Boer, Vincent C; Frezza, Christian; Schulze, Almut; van de Water, Bob; Ruppin, Eytan

    2014-08-01

    Over the last decade, the field of cancer metabolism has mainly focused on studying the role of tumorigenic metabolic rewiring in supporting cancer proliferation. Here, we perform the first genome-scale computational study of the metabolic underpinnings of cancer migration. We build genome-scale metabolic models of the NCI-60 cell lines that capture the Warburg effect (aerobic glycolysis) typically occurring in cancer cells. The extent of the Warburg effect in each of these cell line models is quantified by the ratio of glycolytic to oxidative ATP flux (AFR), which is found to be highly positively associated with cancer cell migration. We hence predicted that targeting genes that mitigate the Warburg effect by reducing the AFR may specifically inhibit cancer migration. By testing the anti-migratory effects of silencing such 17 top predicted genes in four breast and lung cancer cell lines, we find that up to 13 of these novel predictions significantly attenuate cell migration either in all or one cell line only, while having almost no effect on cell proliferation. Furthermore, in accordance with the predictions, a significant reduction is observed in the ratio between experimentally measured ECAR and OCR levels following these perturbations. Inhibiting anti-migratory targets is a promising future avenue in treating cancer since it may decrease cytotoxic-related side effects that plague current anti-proliferative treatments. Furthermore, it may reduce cytotoxic-related clonal selection of more aggressive cancer cells and the likelihood of emerging resistance. © 2014 The Authors. Published under the terms of the CC BY 4.0 license.

  16. Repurposing Approach Identifies Auranofin with Broad Spectrum Antifungal Activity That Targets Mia40-Erv1 Pathway

    Science.gov (United States)

    Thangamani, Shankar; Maland, Matthew; Mohammad, Haroon; Pascuzzi, Pete E.; Avramova, Larisa; Koehler, Carla M.; Hazbun, Tony R.; Seleem, Mohamed N.

    2017-01-01

    Current antifungal therapies have limited effectiveness in treating invasive fungal infections. Furthermore, the development of new antifungal is currently unable to keep pace with the urgent demand for safe and effective new drugs. Auranofin, an FDA-approved drug for the treatment of rheumatoid arthritis, inhibits growth of a diverse array of clinical isolates of fungi and represents a new antifungal agent with a previously unexploited mechanism of action. In addition to auranofin's potent antifungal activity against planktonic fungi, this drug significantly reduces the metabolic activity of Candida cells encased in a biofilm. Unbiased chemogenomic profiling, using heterozygous S. cerevisiae deletion strains, combined with growth assays revealed three probable targets for auranofin's antifungal activity—mia40, acn9, and coa4. Mia40 is of particular interest given its essential role in oxidation of cysteine rich proteins imported into the mitochondria. Biochemical analysis confirmed auranofin targets the Mia40-Erv1 pathway as the drug inhibited Mia40 from interacting with its substrate, Cmc1, in a dose-dependent manner similar to the control, MB-7. Furthermore, yeast mitochondria overexpressing Erv1 were shown to exhibit resistance to auranofin as an increase in Cmc1 import was observed compared to wild-type yeast. Further in vivo antifungal activity of auranofin was examined in a Caenorhabditis elegans animal model of Cryptococcus neoformans infection. Auranofin significantly reduced the fungal load in infected C. elegans. Collectively, the present study provides valuable evidence that auranofin has significant promise to be repurposed as a novel antifungal agent and may offer a safe, effective, and quick supplement to current approaches for treating fungal infections. PMID:28149831

  17. Novel targets of sulforaphane in primary cardiomyocytes identified by proteomic analysis.

    Science.gov (United States)

    Angeloni, Cristina; Turroni, Silvia; Bianchi, Laura; Fabbri, Daniele; Motori, Elisa; Malaguti, Marco; Leoncini, Emanuela; Maraldi, Tullia; Bini, Luca; Brigidi, Patrizia; Hrelia, Silvana

    2013-01-01

    Cardiovascular diseases represent the main cause of mortality in the industrialized world and the identification of effective preventive strategies is of fundamental importance. Sulforaphane, an isothiocyanate from cruciferous vegetables, has been shown to up-regulate phase II enzymes in cardiomyocytes and counteract oxidative stress-induced apoptosis. Aim of the present study was the identification and characterization of novel sulforaphane targets in cardiomyocytes applying a proteomic approach. Two-dimensional gel electrophoresis and mass spectrometry were used to generate protein profiles of primary neonatal rat cardiomyocytes treated and untreated with 5 µM sulforaphane for 1-48 h. According to image analysis, 64 protein spots were found as differentially expressed and their functional correlations were investigated using the MetaCore program. We mainly focused on 3 proteins: macrophage migration inhibitory factor (MIF), CLP36 or Elfin, and glyoxalase 1, due to their possible involvement in cardioprotection. Validation of the time-dependent differential expression of these proteins was performed by western blotting. In particular, to gain insight into the cardioprotective role of the modulation of glyoxalase 1 by sulforaphane, further experiments were performed using methylglyoxal to mimic glycative stress. Sulforaphane was able to counteract methylglyoxal-induced apoptosis, ROS production, and glycative stress, likely through glyoxalase 1 up-regulation. In this study, we reported for the first time new molecular targets of sulforaphane, such as MIF, CLP36 and glyoxalase 1. In particular, we gave new insights into the anti-glycative role of sulforaphane in cardiomyocytes, confirming its pleiotropic behavior in counteracting cardiovascular diseases.

  18. Use of a bacteriophage lysin to identify a novel target for antimicrobial development.

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    Raymond Schuch

    Full Text Available We identified an essential cell wall biosynthetic enzyme in Bacillus anthracis and an inhibitor thereof to which the organism did not spontaneously evolve measurable resistance. This work is based on the exquisite binding specificity of bacteriophage-encoded cell wall-hydrolytic lysins, which have evolved to recognize critical receptors within the bacterial cell wall. Focusing on the B. anthracis-specific PlyG lysin, we first identified its unique cell wall receptor and cognate biosynthetic pathway. Within this pathway, one biosynthetic enzyme, 2-epimerase, was required for both PlyG receptor expression and bacterial growth. The 2-epimerase was used to design a small-molecule inhibitor, epimerox. Epimerox prevented growth of several Gram-positive pathogens and rescued mice challenged with lethal doses of B. anthracis. Importantly, resistance to epimerox was not detected (<10(-11 frequency in B. anthracis and S. aureus. These results describe the use of phage lysins to identify promising lead molecules with reduced resistance potential for antimicrobial development.

  19. Systematic genetic analysis identifies Cis-eQTL target genes associated with glioblastoma patient survival.

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    Qing-Rong Chen

    Full Text Available Prior expression quantitative trait locus (eQTL studies have demonstrated heritable variation determining differences in gene expression. The majority of eQTL studies were based on cell lines and normal tissues. We performed cis-eQTL analysis using glioblastoma multiforme (GBM data sets obtained from The Cancer Genome Atlas (TCGA to systematically investigate germline variation's contribution to tumor gene expression levels. We identified 985 significant cis-eQTL associations (FDR<0.05 mapped to 978 SNP loci and 159 unique genes. Approximately 57% of these eQTLs have been previously linked to the gene expression in cell lines and normal tissues; 43% of these share cis associations known to be associated with functional annotations. About 25% of these cis-eQTL associations are also common to those identified in Breast Cancer from a recent study. Further investigation of the relationship between gene expression and patient clinical information identified 13 eQTL genes whose expression level significantly correlates with GBM patient survival (p<0.05. Most of these genes are also differentially expressed in tumor samples and organ-specific controls (p<0.05. Our results demonstrated a significant relationship of germline variation with gene expression levels in GBM. The identification of eQTLs-based expression associated survival might be important to the understanding of genetic contribution to GBM cancer prognosis.

  20. Identification of a mitochondrial target of thiazolidinedione insulin sensitizers (mTOT--relationship to newly identified mitochondrial pyruvate carrier proteins.

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    Jerry R Colca

    Full Text Available Thiazolidinedione (TZD insulin sensitizers have the potential to effectively treat a number of human diseases, however the currently available agents have dose-limiting side effects that are mediated via activation of the transcription factor PPARγ. We have recently shown PPARγ-independent actions of TZD insulin sensitizers, but the molecular target of these molecules remained to be identified. Here we use a photo-catalyzable drug analog probe and mass spectrometry-based proteomics to identify a previously uncharacterized mitochondrial complex that specifically recognizes TZDs. These studies identify two well-conserved proteins previously known as brain protein 44 (BRP44 and BRP44 Like (BRP44L, which recently have been renamed Mpc2 and Mpc1 to signify their function as a mitochondrial pyruvate carrier complex. Knockdown of Mpc1 or Mpc2 in Drosophila melanogaster or pre-incubation with UK5099, an inhibitor of pyruvate transport, blocks the crosslinking of mitochondrial membranes by the TZD probe. Knockdown of these proteins in Drosophila also led to increased hemolymph glucose and blocked drug action. In isolated brown adipose tissue (BAT cells, MSDC-0602, a PPARγ-sparing TZD, altered the incorporation of (13C-labeled carbon from glucose into acetyl CoA. These results identify Mpc1 and Mpc2 as components of the mitochondrial target of TZDs (mTOT and suggest that understanding the modulation of this complex, which appears to regulate pyruvate entry into the mitochondria, may provide a viable target for insulin sensitizing pharmacology.

  1. Bonafide Targets of Deregulated microRNAs in Non-Small Cell Lung Cancer as Tool to Identify Novel Therapeutic Targets: A Review.

    Science.gov (United States)

    Cipollini, Monica; Landi, Stefano; Gemignani, Federica

    2017-01-01

    Non-small-cell lung cancer (NSCLC) is an aggressive neoplasm with a poor survival and novel therapies are urgently needed. The study of deregulated micro- RNAs (dereg-miRs) could constitute a strategy helping to detect specific genes playing a relevant role in the disease. Thus, the oncoproteins encoded by these genes could be exploited as novel therapeutic targets to be inhibited by small molecules, aptamers, or monoclonal antibodies. The present review is focused on candidate genes having convincing biological evidences to be both bona fide targets for dereg-miRs and playing a role in NSCLC progression. These genes were evaluated according to the molecular pathway they belong. Moreover, in the attempt to provide an even broader list of candidate therapeutic targets for NSCLC, the full list of genes was analyzed using the online tool Interactome DB. Among the identified targets, some of them belong to p53 or MAP kinase signaling pathways, and others include caspases, MCL1, and BCL2L2 (playing a role in apoptosis), ZEB1, ZEB2, and USP25 (epithelial-to-mesenchymal transition), EZH2, SOX9, and HOXA5 (differentiation), Paxillin, LIMK1 and MTDH (cytoskeleton remodeling), and HDGF (angiogenesis). In addition, other targets, such as TIMP-2, PIM-1, and components of the IGF-signaling pathways were suggested following the interactome analysis. Studies on dereg-miRs helped to identify a set of genes whose encoded proteins could constitute candidates for future therapeutic approaches. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  2. Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci.

    Science.gov (United States)

    Sazonova, Olga; Zhao, Yuqi; Nürnberg, Sylvia; Miller, Clint; Pjanic, Milos; Castano, Victor G; Kim, Juyong B; Salfati, Elias L; Kundaje, Anshul B; Bejerano, Gill; Assimes, Themistocles; Yang, Xia; Quertermous, Thomas

    2015-05-01

    To functionally link coronary artery disease (CAD) causal genes identified by genome wide association studies (GWAS), and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq) with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC). Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including "growth factor binding," "matrix interaction," and "smooth muscle contraction." We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.

  3. Characterization of TCF21 Downstream Target Regions Identifies a Transcriptional Network Linking Multiple Independent Coronary Artery Disease Loci.

    Directory of Open Access Journals (Sweden)

    Olga Sazonova

    2015-05-01

    Full Text Available To functionally link coronary artery disease (CAD causal genes identified by genome wide association studies (GWAS, and to investigate the cellular and molecular mechanisms of atherosclerosis, we have used chromatin immunoprecipitation sequencing (ChIP-Seq with the CAD associated transcription factor TCF21 in human coronary artery smooth muscle cells (HCASMC. Analysis of identified TCF21 target genes for enrichment of molecular and cellular annotation terms identified processes relevant to CAD pathophysiology, including "growth factor binding," "matrix interaction," and "smooth muscle contraction." We characterized the canonical binding sequence for TCF21 as CAGCTG, identified AP-1 binding sites in TCF21 peaks, and by conducting ChIP-Seq for JUN and JUND in HCASMC confirmed that there is significant overlap between TCF21 and AP-1 binding loci in this cell type. Expression quantitative trait variation mapped to target genes of TCF21 was significantly enriched among variants with low P-values in the GWAS analyses, suggesting a possible functional interaction between TCF21 binding and causal variants in other CAD disease loci. Separate enrichment analyses found over-representation of TCF21 target genes among CAD associated genes, and linkage disequilibrium between TCF21 peak variation and that found in GWAS loci, consistent with the hypothesis that TCF21 may affect disease risk through interaction with other disease associated loci. Interestingly, enrichment for TCF21 target genes was also found among other genome wide association phenotypes, including height and inflammatory bowel disease, suggesting a functional profile important for basic cellular processes in non-vascular tissues. Thus, data and analyses presented here suggest that study of GWAS transcription factors may be a highly useful approach to identifying disease gene interactions and thus pathways that may be relevant to complex disease etiology.

  4. An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

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    Jonathan A Scolnick

    Full Text Available Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET, for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE tissue RNA in both normal tissue and cancer cells.

  5. An Efficient Method for Identifying Gene Fusions by Targeted RNA Sequencing from Fresh Frozen and FFPE Samples.

    Science.gov (United States)

    Scolnick, Jonathan A; Dimon, Michelle; Wang, I-Ching; Huelga, Stephanie C; Amorese, Douglas A

    2015-01-01

    Fusion genes are known to be key drivers of tumor growth in several types of cancer. Traditionally, detecting fusion genes has been a difficult task based on fluorescent in situ hybridization to detect chromosomal abnormalities. More recently, RNA sequencing has enabled an increased pace of fusion gene identification. However, RNA-Seq is inefficient for the identification of fusion genes due to the high number of sequencing reads needed to detect the small number of fusion transcripts present in cells of interest. Here we describe a method, Single Primer Enrichment Technology (SPET), for targeted RNA sequencing that is customizable to any target genes, is simple to use, and efficiently detects gene fusions. Using SPET to target 5701 exons of 401 known cancer fusion genes for sequencing, we were able to identify known and previously unreported gene fusions from both fresh-frozen and formalin-fixed paraffin-embedded (FFPE) tissue RNA in both normal tissue and cancer cells.

  6. Systems biology and longevity: an emerging approach to identify innovative anti-aging targets and strategies.

    Science.gov (United States)

    Cevenini, E; Bellavista, E; Tieri, P; Castellani, G; Lescai, F; Francesconi, M; Mishto, M; Santoro, A; Valensin, S; Salvioli, S; Capri, M; Zaikin, A; Monti, D; de Magalhães, J P; Franceschi, C

    2010-01-01

    Human aging and longevity are complex and multi-factorial traits that result from a combination of environmental, genetic, epigenetic and stochastic factors, each contributing to the overall phenotype. The multi-factorial process of aging acts at different levels of complexity, from molecule to cell, from organ to organ systems and finally to organism, giving rise to the dynamic "aging mosaic". At present, an increasing amount of experimental data on genetics, genomics, proteomics and other -omics are available thanks to new high-throughput technologies but a comprehensive model for the study of human aging and longevity is still lacking. Systems biology represents a strategy to integrate and quantify the existing knowledge from different sources into predictive models, to be later tested and then implemented with new experimental data for validation and refinement in a recursive process. The ultimate goal is to compact the new acquired knowledge into a single picture, ideally able to characterize the phenotype at systemic/organism level. In this review we will briefly discuss the aging phenotype in a systems biology perspective, showing four specific examples at different levels of complexity, from a systemic process (inflammation) to a cascade-process pathways (coagulation) and from cellular organelle (proteasome) to single gene-network (PON-1), which could also represent targets for anti-aging strategies.

  7. The neurobiology of bipolar disorder: identifying targets for specific agents and synergies for combination treatment.

    Science.gov (United States)

    Andreazza, Ana C; Young, L Trevor

    2014-07-01

    Bipolar disorder (BD) is a chronic psychiatric illness described by severe changes in mood. Extensive research has been carried out to understand the aetiology and pathophysiology of BD. Several hypotheses have been postulated, including alteration in genetic factors, protein expression, calcium signalling, neuropathological alteration, mitochondrial dysfunction and oxidative stress in BD. In the following paper, we will attempt to integrate these data in a manner which is to understand targets of treatment and how they may be, in particular, relevant to combination treatment. In summary, the data suggested that BD might be associated with neuronal and glial cellular impairment in specific brain areas, including the prefrontal cortex. From molecular and genetics: (1) alterations in dopaminergic system, through catechol-O-aminotransferase; (2) decreased expression and polymorphism on brain-derived neurotrophic factor; (3) alterations cyclic-AMP responsive element binding; (4) dysregulation of calcium signalling, including genome-wide finding for voltage-dependent calcium channel α-1 subunit are relevant findings in BD. Future studies are now necessary to understand how these molecular pathways interact and their connection to the complex clinical manifestations observed in BD.

  8. Simultaneous SPECT imaging of multi-targets to assist in identifying hepatic lesions

    Science.gov (United States)

    Guo, Zhide; Gao, Mengna; Zhang, Deliang; Li, Yesen; Song, Manli; Zhuang, Rongqiang; Su, Xinhui; Chen, Guibing; Liu, Ting; Liu, Pingguo; Wu, Hua; Du, Jin; Zhang, Xianzhong

    2016-01-01

    Molecular imaging technique is an attractive tool to detect liver disease at early stage. This study aims to develop a simultaneous dual-isotope single photon emission computed tomography (SPECT)/CT imaging method to assist diagnosis of hepatic tumor and liver fibrosis. Animal models of liver fibrosis and orthotopic human hepatocellular carcinoma (HCC) were established. The tracers of 131I-NGA and 99mTc-3P-RGD2 were selected to target asialoglycoprotein receptor (ASGPR) on the hepatocytes and integrin αvβ3 receptor in tumor or fibrotic liver, respectively. SPECT imaging and biodistribution study were carried out to verify the feasibility and superiority. As expected, 99mTc-3P-RGD2 had the ability to evaluate liver fibrosis and detect tumor lesions. 131I-NGA showed that it was effective in assessing the anatomy and function of the liver. In synchronized dual-isotope SPECT/CT imaging, clear fusion images can be got within 30 minutes for diagnosing liver fibrosis and liver cancer. This new developed imaging approach enables the acquisition of different physiological information for diagnosing liver fibrosis, liver cancer and evaluating residual functional liver volume simultaneously. So synchronized dual-isotope SPECT/CT imaging with 99mTc-3P-RGD2 and 131I-NGA is an effective approach to detect liver disease, especially liver fibrosis and liver cancer. PMID:27377130

  9. Identifying undetected cases of chronic kidney disease in Mexico. Targeting high-risk populations.

    Science.gov (United States)

    García-García, Guillermo; Gutiérrez-Padilla, Alfonso J; Chávez-Iñiguez, Jonathan; Pérez-Gómez, Héctor R; Mendoza-García, Martha; González-De la Peña, Ma Del Mar; Tonelli, Marcello

    2013-11-01

    Chronic kidney disease (CKD) is a major public health problem in Mexico. Current guidelines recommend routine CKD testing in patients at increased risk for CKD. We undertook this study to examine the diagnostic yield of targeted screening (case-finding) for CKD in high-risk populations in rural and urban communities in Jalisco, Mexico. In a cross-sectional study, we did laboratory tests searching for CKD and its risk factors and compared the characteristics of participants with those reported by the National Health and Nutrition Survey 2006 (NHNS). Individuals who were aware that they had CKD and those 126 mg/dl (56.1 vs. 14.4%, p = 0.0001) were more prevalent among the participants; self-reported hypertension (41.9 vs. 7.3%, p = 0.0001), systolic blood pressure ≥ 140 mm Hg or diastolic blood pressure ≥ 90 mm Hg (52.5 vs. 43.2%, p = 0.0001), and obesity (42.8. vs. 29.3%, p = 0.0001) were also more frequent among participants. There were 19.7% with proteinuria; CKD was more prevalent among the high risk participants in our study (31.3 vs. 8.0%, p = 0.0001) than in the general population. CKD was detected frequently in high-risk Mexican populations. Trials of case-finding and intervention are feasible and warranted in Mexico. Copyright © 2013 IMSS. Published by Elsevier Inc. All rights reserved.

  10. IdealKnock: A framework for efficiently identifying knockout strategies leading to targeted overproduction.

    Science.gov (United States)

    Gu, Deqing; Zhang, Cheng; Zhou, Shengguo; Wei, Liujing; Hua, Qiang

    2016-04-01

    In recent years, computer aided redesigning methods based on genome-scale metabolic network models (GEMs) have played important roles in metabolic engineering studies; however, most of these methods are hindered by intractable computing times. In particular, methods that predict knockout strategies leading to overproduction of desired biochemical are generally unable to do high level prediction because the computational time will increase exponentially. In this study, we propose a new framework named IdealKnock, which is able to efficiently evaluate potentials of the production for different biochemical in a system by merely knocking out pathways. In addition, it is also capable of searching knockout strategies when combined with the OptKnock or OptGene framework. Furthermore, unlike other methods, IdealKnock suggests a series of mutants with targeted overproduction, which enables researchers to select the one of greatest interest for experimental validation. By testing the overproduction of a large number of native metabolites, IdealKnock showed its advantage in successfully breaking through the limitation of maximum knockout number in reasonable time and suggesting knockout strategies with better performance than other methods. In addition, gene-reaction relationship is well considered in the proposed framework.

  11. Carbohydrate Microarrays Identify Blood Group Precursor Cryptic Epitopes as Potential Immunological Targets of Breast Cancer

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    Denong Wang

    2015-01-01

    Full Text Available Using carbohydrate microarrays, we explored potential natural ligands of antitumor monoclonal antibody HAE3. This antibody was raised against a murine mammary tumor antigen but was found to cross-react with a number of human epithelial tumors in tissues. Our carbohydrate microarray analysis reveals that HAE3 is specific for an O-glycan cryptic epitope that is normally hidden in the cores of blood group substances. Using HAE3 to screen tumor cell surface markers by flow cytometry, we found that the HAE3 glycoepitope, gpHAE3, was highly expressed by a number of human breast cancer cell lines, including some triple-negative cancers that lack the estrogen, progesterone, and Her2/neu receptors. Taken together, we demonstrate that HAE3 recognizes a conserved cryptic glycoepitope of blood group precursors, which is nevertheless selectively expressed and surface-exposed in certain breast tumor cells. The potential of this class of O-glycan cryptic antigens in breast cancer subtyping and targeted immunotherapy warrants further investigation.

  12. Quantitative Phosphoproteomics Identifies Filaggrin and other Targets of Ionizing Radiation in a Human Skin Model

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Feng; Waters, Katrina M.; Webb-Robertson, Bobbie-Jo M.; Sowa, Marianne B.; Freiin von Neubeck, Claere H.; Aldrich, Joshua T.; Markillie, Lye Meng; Wirgau, Rachel M.; Gristenko, Marina A.; Zhao, Rui; Camp, David G.; Smith, Richard D.; Stenoien, David L.

    2012-04-17

    Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low and high dose ionizing radiation dependent signaling in a complex 3-dimensional setting. Application of an isobaric labeling strategy using sham and 3 radiation doses (3, 10, 200 cGy) resulted in the identification of 1113 unique phosphopeptides. Statistical analyses identified 151 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1 and 2,) had altered phosphorylation levels following exposure to both low and high doses of radiation. A phosphorylation site present in multiple copies in the linker regions of human profilaggrin underwent the largest fold change. Increased phosphorylation of these sites coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain skin barrier functions and minimize the damaging consequences of radiation exposure.

  13. Quantitative phosphoproteomics identifies filaggrin and other targets of ionizing radiation in a human skin model.

    Science.gov (United States)

    Yang, Feng; Waters, Katrina M; Webb-Robertson, Bobbie-Jo; Sowa, Marianne B; von Neubeck, Claere; Aldrich, Josh T; Markillie, Lye Meng; Wirgau, Rachel M; Gritsenko, Marina A; Zhao, Rui; Camp, David G; Smith, Richard D; Stenoien, David L

    2012-05-01

    Our objective here was to perform a quantitative phosphoproteomic study on a reconstituted human skin tissue to identify low- and high-dose ionizing radiation-dependent signalling in a complex three-dimensional setting. Application of an isobaric labelling strategy using sham and three radiation doses (3, 10, 200 cGy) resulted in the identification of 1052 unique phosphopeptides. Statistical analyses identified 176 phosphopeptides showing significant changes in response to radiation and radiation dose. Proteins responsible for maintaining skin structural integrity including keratins and desmosomal proteins (desmoglein, desmoplakin, plakophilin 1, 2 and 3) had altered phosphorylation levels following exposure to both low and high doses of radiation. Altered phosphorylation of multiple sites in profilaggrin linker domains coincided with altered profilaggrin processing suggesting a role for linker phosphorylation in human profilaggrin regulation. These studies demonstrate that the reconstituted human skin system undergoes a coordinated response to both low and high doses of ionizing radiation involving multiple layers of the stratified epithelium that serve to maintain tissue integrity and mitigate effects of radiation exposure.

  14. Genetic profile for suspected dysferlinopathy identified by targeted next-generation sequencing

    Science.gov (United States)

    Izumi, Rumiko; Niihori, Tetsuya; Takahashi, Toshiaki; Suzuki, Naoki; Tateyama, Maki; Watanabe, Chigusa; Sugie, Kazuma; Nakanishi, Hirotaka; Sobue, Gen; Kato, Masaaki; Warita, Hitoshi; Aoki, Yoko

    2015-01-01

    Objective: To investigate the genetic causes of suspected dysferlinopathy and to reveal the genetic profile for myopathies with dysferlin deficiency. Methods: Using next-generation sequencing, we analyzed 42 myopathy-associated genes, including DYSF, in 64 patients who were clinically or pathologically suspected of having dysferlinopathy. Putative pathogenic mutations were confirmed by Sanger sequencing. In addition, copy-number variations in DYSF were investigated using multiplex ligation-dependent probe amplification. We also analyzed the genetic profile for 90 patients with myopathy with dysferlin deficiency, as indicated by muscle specimen immunohistochemistry, including patients from a previous cohort. Results: We identified putative pathogenic mutations in 38 patients (59% of all investigated patients). Twenty-three patients had DYSF mutations, including 6 novel mutations. The remaining 16 patients, including a single patient who also carried the DYSF mutation, harbored putative pathogenic mutations in other genes. The genetic profile for 90 patients with dysferlin deficiency revealed that 70% had DYSF mutations (n = 63), 10% had CAPN3 mutations (n = 9), 2% had CAV3 mutations (n = 2), 3% had mutations in other genes (in single patients), and 16% did not have any identified mutations (n = 14). Conclusions: This study clarified the heterogeneous genetic profile for myopathies with dysferlin deficiency. Our results demonstrate the importance of a comprehensive analysis of related genes in improving the genetic diagnosis of dysferlinopathy as one of the most common subtypes of limb-girdle muscular dystrophy. Unresolved diagnoses should be investigated using whole-genome or whole-exome sequencing. PMID:27066573

  15. Chemical Genomics Identifies the PERK-Mediated Unfolded Protein Stress Response as a Cellular Target for Influenza Virus Inhibition

    Directory of Open Access Journals (Sweden)

    Sara Landeras-Bueno

    2016-04-01

    Full Text Available Influenza A viruses generate annual epidemics and occasional pandemics of respiratory disease with important consequences for human health and the economy. Therefore, a large effort has been devoted to the development of new anti-influenza virus drugs directed to viral targets, as well as to the identification of cellular targets amenable to anti-influenza virus therapy. Here we have addressed the identification of such potential cellular targets by screening collections of drugs approved for human use. We reasoned that screening with a green fluorescent protein-based recombinant replicon system would identify cellular targets involved in virus transcription/replication and/or gene expression and hence address an early stage of virus infection. By using such a strategy, we identified Montelukast (MK as an inhibitor of virus multiplication. MK inhibited virus gene expression but did not alter viral RNA synthesis in vitro or viral RNA accumulation in vivo. The low selectivity index of MK prevented its use as an antiviral, but it was sufficient to identify a new cellular pathway suitable for anti-influenza virus intervention. By deep sequencing of RNA isolated from mock- and virus-infected human cells, treated with MK or left untreated, we showed that it stimulates the PERK-mediated unfolded protein stress response. The phosphorylation of PERK was partly inhibited in virus-infected cells but stimulated in MK-treated cells. Accordingly, pharmacological inhibition of PERK phosphorylation led to increased viral gene expression, while inhibition of PERK phosphatase reduced viral protein synthesis. These results suggest the PERK-mediated unfolded protein response as a potential cellular target to modulate influenza virus infection.

  16. Combining phenotypic and proteomic approaches to identify membrane targets in a ‘triple negative’ breast cancer cell type

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    Rust Steven

    2013-02-01

    Full Text Available Abstract Background The continued discovery of therapeutic antibodies, which address unmet medical needs, requires the continued discovery of tractable antibody targets. Multiple protein-level target discovery approaches are available and these can be used in combination to extensively survey relevant cell membranomes. In this study, the MDA-MB-231 cell line was selected for membranome survey as it is a ‘triple negative’ breast cancer cell line, which represents a cancer subtype that is aggressive and has few treatment options. Methods The MDA-MB-231 breast carcinoma cell line was used to explore three membranome target discovery approaches, which were used in parallel to cross-validate the significance of identified antigens. A proteomic approach, which used membrane protein enrichment followed by protein identification by mass spectrometry, was used alongside two phenotypic antibody screening approaches. The first phenotypic screening approach was based on hybridoma technology and the second was based on phage display technology. Antibodies isolated by the phenotypic approaches were tested for cell specificity as well as internalisation and the targets identified were compared to each other as well as those identified by the proteomic approach. An anti-CD73 antibody derived from the phage display-based phenotypic approach was tested for binding to other ‘triple negative’ breast cancer cell lines and tested for tumour growth inhibitory activity in a MDA-MB-231 xenograft model. Results All of the approaches identified multiple cell surface markers, including integrins, CD44, EGFR, CD71, galectin-3, CD73 and BCAM, some of which had been previously confirmed as being tractable to antibody therapy. In total, 40 cell surface markers were identified for further study. In addition to cell surface marker identification, the phenotypic antibody screening approaches provided reagent antibodies for target validation studies. This is illustrated

  17. Target Mechanism-Based Whole-Cell Screening Identifies Bortezomib as an Inhibitor of Caseinolytic Protease in Mycobacteria

    Science.gov (United States)

    Moreira, Wilfried; Ngan, Grace J. Y.; Low, Jian Liang; Poulsen, Anders; Chia, Brian C. S.; Ang, Melgious J. Y.; Yap, Amelia; Fulwood, Justina; Lakshmanan, Umayal; Lim, Jolander; Khoo, Audrey Y. T.; Flotow, Horst; Hill, Jeffrey; Raju, Ravikiran M.; Rubin, Eric J.

    2015-01-01

    ABSTRACT A novel type of antibacterial screening method, a target mechanism-based whole-cell screening method, was developed to combine the advantages of target mechanism- and whole-cell-based approaches. A mycobacterial reporter strain with a synthetic phenotype for caseinolytic protease (ClpP1P2) activity was engineered, allowing the detection of inhibitors of this enzyme inside intact bacilli. A high-throughput screening method identified bortezomib, a human 26S proteasome drug, as a potent inhibitor of ClpP1P2 activity and bacterial growth. A battery of secondary assays was employed to demonstrate that bortezomib indeed exerts its antimicrobial activity via inhibition of ClpP1P2: Down- or upmodulation of the intracellular protease level resulted in hyper- or hyposensitivity of the bacteria, the drug showed specific potentiation of translation error-inducing aminoglycosides, ClpP1P2-specific substrate WhiB1 accumulated upon exposure, and growth inhibition potencies of bortezomib derivatives correlated with ClpP1P2 inhibition potencies. Furthermore, molecular modeling showed that the drug can bind to the catalytic sites of ClpP1P2. This work demonstrates the feasibility of target mechanism-based whole-cell screening, provides chemical validation of ClpP1P2 as a target, and identifies a drug in clinical use as a new lead compound for tuberculosis therapy. PMID:25944857

  18. Identifying New Drug Targets for Potent Phospholipase D Inhibitors: Combining Sequence Alignment, Molecular Docking, and Enzyme Activity/Binding Assays.

    Science.gov (United States)

    Djakpa, Helene; Kulkarni, Aditya; Barrows-Murphy, Scheneque; Miller, Greg; Zhou, Weihong; Cho, Hyejin; Török, Béla; Stieglitz, Kimberly

    2016-05-01

    Phospholipase D enzymes cleave phospholipid substrates generating choline and phosphatidic acid. Phospholipase D from Streptomyces chromofuscus is a non-HKD (histidine, lysine, and aspartic acid) phospholipase D as the enzyme is more similar to members of the diverse family of metallo-phosphodiesterase/phosphatase enzymes than phospholipase D enzymes with active site HKD repeats. A highly efficient library of phospholipase D inhibitors based on 1,3-disubstituted-4-amino-pyrazolopyrimidine core structure was utilized to evaluate the inhibition of purified S. chromofuscus phospholipase D. The molecules exhibited inhibition of phospholipase D activity (IC50 ) in the nanomolar range with monomeric substrate diC4 PC and micromolar range with phospholipid micelles and vesicles. Binding studies with vesicle substrate and phospholipase D strongly indicate that these inhibitors directly block enzyme vesicle binding. Following these compelling results as a starting point, sequence searches and alignments with S. chromofuscus phospholipase D have identified potential new drug targets. Using AutoDock, inhibitors were docked into the enzymes selected from sequence searches and alignments (when 3D co-ordinates were available) and results analyzed to develop next-generation inhibitors for new targets. In vitro enzyme activity assays with several human phosphatases demonstrated that the predictive protocol was accurate. The strategy of combining sequence comparison, docking, and high-throughput screening assays has helped to identify new drug targets and provided some insight into how to make potential inhibitors more specific to desired targets.

  19. Phosphotyrosine profiling identifies ephrin receptor A2 as a potential therapeutic target in esophageal squamous-cell carcinoma.

    Science.gov (United States)

    Syed, Nazia; Barbhuiya, Mustafa A; Pinto, Sneha M; Nirujogi, Raja Sekhar; Renuse, Santosh; Datta, Keshava K; Khan, Aafaque Ahmad; Srikumar, Kotteazeth; Prasad, T S Keshava; Kumar, M Vijaya; Kumar, Rekha Vijay; Chatterjee, Aditi; Pandey, Akhilesh; Gowda, Harsha

    2015-01-01

    Esophageal squamous-cell carcinoma (ESCC) is one of the most common malignancies in Asia. Currently, surgical resection of early-stage tumor is the best available treatment. However, most patients present late when surgery is not an option. Data suggest that chemotherapy regimens are inadequate for clinical management of advanced cancer. Targeted therapy has emerged as one of the most promising approaches to treat several malignancies. A prerequisite for developing targeted therapy is prior knowledge of proteins and pathways that drive proliferation in malignancies. We carried out phosphotyrosine profiling across four different ESCC cell lines and compared it to non-neoplastic Het-1A cell line to identify activated tyrosine kinase signaling pathways in ESCC. A total of 278 unique phosphopeptides were identified across these cell lines. This included several tyrosine kinases and their substrates that were hyperphosphorylated in ESCC. Ephrin receptor A2 (EPHA2), a receptor tyrosine kinase, was hyperphosphorylated in all the ESCC cell lines used in the study. EPHA2 is reported to be oncogenic in several cancers and is also known to promote metastasis. Immunohistochemistry-based studies have revealed EPHA2 is overexpressed in nearly 50% of ESCC. We demonstrated EPHA2 as a potential therapeutic target in ESCC by carrying out siRNA-based knockdown studies. Knockdown of EPHA2 in ESCC cell line TE8 resulted in significant decrease in cell proliferation and invasion, suggesting it is a promising therapeutic target in ESCC that warrants further evaluation.

  20. Microarray analyses of SREBP-1a and SREBP-1c target genes identify new regulatory pathways in muscle.

    OpenAIRE

    Rome, Sophie; Lecomte, Virginie; Meugnier, Emmanuelle; Rieusset, Jennifer; Debard, Cyrille; Euthine, Vanessa; Vidal, Hubert; Lefai, Etienne

    2008-01-01

    International audience; In this study we have identified the target genes of sterol regulatory element binding protein (SREBP)-1a and SREBP-1c in primary cultures of human skeletal muscle cells, using adenoviral vectors expressing the mature nuclear form of human SREBP-1a or SREBP-1c combined with oligonucleotide microarrays. Overexpression of SREBP-1a led to significant changes in the expression of 1,315 genes (655 upregulated and 660 downregulated), whereas overexpression of SREBP-1c modifi...

  1. The Study and Prioritization of the Role of Market Segmentation Criteria in Identifying Bank Target Market

    Directory of Open Access Journals (Sweden)

    Siavash Rashidi

    2013-05-01

    Full Text Available Banks have today become the world's main economic development and trade exchanges. So that, without efficient and up to date banks is virtually impossible to achieve economic prosperity. Bank is an undeniable economic infrastructure and in these circumstances, the bank is also ending the competition, they are forced to develop new methods of banking. Given the current business environment, it is likely that market segmentation to be considered a key strategic issue for service providers. In more developed economies, the importance of market segmentation has been well documented in the financial services sector. When the competition is fierce in the banking market, financial services organizations is largely adopt a strategy based on segmentation complex shapes. The main objective of this study was to investigate the role of market segmentation criteria on the identification and prioritization of banks target markets. Applied research method, is descriptive and correlation research; which field research and library studies were used for data collection. Given that, the population in this study was a sampling from all branches of Refah Bank in Zanjan city, data and information collected from them conducted through questionnaires and were tested using statistical software Spss18. To assess the presence or absence of the relationship between variables and segmentation, Correlation test was used. Thus, the priority of each of these factors from the perspective Bank customers was obtained by the Friedman test. Also, using the correlation test, relationship between psychological variables and behavior of the customers were calculated and investigated with the main factors of bank market.Our results indicate that the "human" factor have more important and the "incentives" have less importance in preferred standards of banking services from the perspective of bank customers in Zanjan city, also it has been demonstrated that there is a significant

  2. Identifying Genetic Sources of Phenotypic Heterogeneity in Orofacial Clefts by Targeted Sequencing.

    Science.gov (United States)

    Carlson, Jenna C; Taub, Margaret A; Feingold, Eleanor; Beaty, Terri H; Murray, Jeffrey C; Marazita, Mary L; Leslie, Elizabeth J

    2017-07-17

    Orofacial clefts (OFCs), including nonsyndromic cleft lip with or without cleft palate (NSCL/P), are common birth defects. NSCL/P is highly heterogeneous with multiple phenotypic presentations. Two common subtypes of NSCL/P are cleft lip (CL) and cleft lip with cleft palate (CLP) which have different population prevalence. Similarly, NSCL/P can be divided into bilateral and unilateral clefts, with unilateral being the most common. Individuals with unilateral NSCL/P are more likely to be affected on the left side of the upper lip, but right side affection also occurs. Moreover, NSCL/P is twice as common in males as in females. The goal of this study is to discover genetic variants that have different effects in case subgroups. We conducted both common variant and rare variant analyses in 1034 individuals of Asian ancestry with NSCL/P, examining four sources of heterogeneity within CL/P: cleft type, sex, laterality, and side. We identified several regions associated with subtype differentiation: cleft type differences in 8q24 (p = 1.00 × 10(-4) ), laterality differences in IRF6, a gene previously implicated with wound healing (p = 2.166 × 10(-4) ), sex differences and side of unilateral CL differences in FGFR2 (p = 3.00 × 10(-4) ; p = 6.00 × 10(-4) ), and sex differences in VAX1 (p < 1.00 × 10(-4) ) among others. Many of the regions associated with phenotypic modification were either adjacent to or overlapping functional elements based on ENCODE chromatin marks and published craniofacial enhancers. We have identified multiple common and rare variants as potential phenotypic modifiers of NSCL/P, and suggest plausible elements responsible for phenotypic heterogeneity, further elucidating the complex genetic architecture of OFCs. Birth Defects Research 109:1030-1038, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

  3. A Comparative Reverse Docking Strategy to Identify Potential Antineoplastic Targets of Tea Functional Components and Binding Mode

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    Rong Zheng

    2011-08-01

    Full Text Available The main functional components of green tea, such as epigallocatechin gallate (EGCG, epigallocatechin (EGC, epicatechin gallate (ECG and epicatechin (EC, are found to have a broad antineoplastic activity. The discovery of their targets plays an important role in revealing the antineoplastic mechanism. Therefore, to identify potential target proteins for tea polyphenols, we have taken a comparative virtual screening approach using two reverse docking systems, one based on Autodock software and the other on Tarfisdock. Two separate in silico workflows were implemented to derive a set of target proteins related to human diseases and ranked by the binding energy score. Several conventional clinically important proteins with anti-tumor effects are screened out from the PDTD protein database as the potential receptors by both procedures. To further analyze the validity of docking results, we study the binding mode of EGCG and the potential target protein Leukotriene A4 hydrolase in detail. We indicate that interactions mediated by electrostatic and hydrogen bond play a key role in ligand binding. EGCG binds to the enzyme with certain orientation and conformation that is suitable for nucleophilic attacks by several electrical residues inside the enzyme’s activity cavity. This study provides useful information for studying the antitumor mechanism of tea’s functional components. The comparative reverse docking strategy presented generates a tractable set of antineoplastic proteins for future experimental validation as drug targets against tumors.

  4. Targeting N-Glycan Cryptic Sugar Moieties for Broad-Spectrum Virus Neutralization: Progress in Identifying Conserved Molecular Targets in Viruses of Distinct Phylogenetic Origins

    Directory of Open Access Journals (Sweden)

    Denong Wang

    2015-03-01

    Full Text Available Identifying molecular targets for eliciting broadly virus-neutralizing antibodies is one of the key steps toward development of vaccines against emerging viral pathogens. Owing to genomic and somatic diversities among viral species, identifying protein targets for broad-spectrum virus neutralization is highly challenging even for the same virus, such as HIV-1. However, viruses rely on host glycosylation machineries to synthesize and express glycans and, thereby, may display common carbohydrate moieties. Thus, exploring glycan-binding profiles of broad-spectrum virus-neutralizing agents may provide key information to uncover the carbohydrate-based virus-neutralizing epitopes. In this study, we characterized two broadly HIV-neutralizing agents, human monoclonal antibody 2G12 and Galanthus nivalis lectin (GNA, for their viral targeting activities. Although these agents were known to be specific for oligomannosyl antigens, they differ strikingly in virus-binding activities. The former is HIV-1 specific; the latter is broadly reactive and is able to neutralize viruses of distinct phylogenetic origins, such as HIV-1, severe acute respiratory syndrome coronavirus (SARS-CoV, and human cytomegalovirus (HCMV. In carbohydrate microarray analyses, we explored the molecular basis underlying the striking differences in the spectrum of anti-virus activities of the two probes. Unlike 2G12, which is strictly specific for the high-density Man9GlcNAc2Asn (Man9-clusters, GNA recognizes a number of N-glycan cryptic sugar moieties. These include not only the known oligomannosyl antigens but also previously unrecognized tri-antennary or multi-valent GlcNAc-terminating N-glycan epitopes (Tri/m-Gn. These findings highlight the potential of N-glycan cryptic sugar moieties as conserved targets for broad-spectrum virus neutralization and suggest the GNA-model of glycan-binding warrants focused investigation.

  5. Transcriptome Analysis Identifies the Dysregulation of Ultraviolet Target Genes in Human Skin Cancers.

    Science.gov (United States)

    Shen, Yao; Kim, Arianna L; Du, Rong; Liu, Liang

    2016-01-01

    Exposure to ultraviolet radiation (UVR) is a major risk factor for both melanoma and non-melanoma skin cancers. In addition to its mutagenic effect, UVR can also induce substantial transcriptional instability in skin cells affecting thousands of genes, including many cancer genes, suggesting that transcriptional instability may be another important etiological factor in skin photocarcinogenesis. In this study, we performed detailed transcriptomic profiling studies to characterize the kinetic changes in global gene expression in human keratinocytes exposed to different UVR conditions. We identified a subset of UV-responsive genes as UV signature genes (UVSGs) based on 1) conserved UV-responsiveness of this subset of genes among different keratinocyte lines; and 2) UV-induced persistent changes in their mRNA levels long after exposure. Interestingly, 11 of the UVSGs were shown to be critical to skin cancer cell proliferation and survival. Through computational Gene Set Enrichment Analysis, we demonstrated that a significant portion of the UVSGs were dysregulated in human skin squamous cell carcinomas, but not in other human malignancies. This highlights the potential and specificity of the UVSGs in clinical diagnosis of UV damage and stratification of skin cancer risk.

  6. Gene expression profiling identifies molecular pathways associated with collagen VI deficiency and provides novel therapeutic targets.

    Directory of Open Access Journals (Sweden)

    Sonia Paco

    Full Text Available Ullrich congenital muscular dystrophy (UCMD, caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered.

  7. Gene Expression Profiling Identifies Molecular Pathways Associated with Collagen VI Deficiency and Provides Novel Therapeutic Targets

    Science.gov (United States)

    Paco, Sonia; Kalko, Susana G.; Jou, Cristina; Rodríguez, María A.; Corbera, Joan; Muntoni, Francesco; Feng, Lucy; Rivas, Eloy; Torner, Ferran; Gualandi, Francesca; Gomez-Foix, Anna M.; Ferrer, Anna; Ortez, Carlos; Nascimento, Andrés; Colomer, Jaume; Jimenez-Mallebrera, Cecilia

    2013-01-01

    Ullrich congenital muscular dystrophy (UCMD), caused by collagen VI deficiency, is a common congenital muscular dystrophy. At present, the role of collagen VI in muscle and the mechanism of disease are not fully understood. To address this we have applied microarrays to analyse the transcriptome of UCMD muscle and compare it to healthy muscle and other muscular dystrophies. We identified 389 genes which are differentially regulated in UCMD relative to controls. In addition, there were 718 genes differentially expressed between UCMD and dystrophin deficient muscle. In contrast, only 29 genes were altered relative to other congenital muscular dystrophies. Changes in gene expression were confirmed by real-time PCR. The set of regulated genes was analysed by Gene Ontology, KEGG pathways and Ingenuity Pathway analysis to reveal the molecular functions and gene networks associated with collagen VI defects. The most significantly regulated pathways were those involved in muscle regeneration, extracellular matrix remodelling and inflammation. We characterised the immune response in UCMD biopsies as being mainly mediated via M2 macrophages and the complement pathway indicating that anti-inflammatory treatment may be beneficial to UCMD as for other dystrophies. We studied the immunolocalisation of ECM components and found that biglycan, a collagen VI interacting proteoglycan, was reduced in the basal lamina of UCMD patients. We propose that biglycan reduction is secondary to collagen VI loss and that it may be contributing towards UCMD pathophysiology. Consequently, strategies aimed at over-expressing biglycan and restore the link between the muscle cell surface and the extracellular matrix should be considered. PMID:24223098

  8. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

    Science.gov (United States)

    Cheng, Feixiong; Murray, James L; Zhao, Junfei; Sheng, Jinsong; Zhao, Zhongming; Rubin, Donald H

    2016-09-01

    Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

  9. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis.

    Directory of Open Access Journals (Sweden)

    Feixiong Cheng

    2016-09-01

    Full Text Available Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase. Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline that may be potential for antiviral indication (e.g. anti-Ebola. In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics.

  10. Silent genetic alterations identified by targeted next-generation sequencing in pheochromocytoma/paraganglioma: A clinicopathological correlations.

    Science.gov (United States)

    Pillai, Suja; Gopalan, Vinod; Lo, Chung Y; Liew, Victor; Smith, Robert A; Lam, Alfred King Y

    2017-02-01

    The goal of this pilot study was to develop a customized, cost-effective amplicon panel (Ampliseq) for target sequencing in a cohort of patients with sporadic phaeochromocytoma/paraganglioma. Phaeochromocytoma/paragangliomas from 25 patients were analysed by targeted next-generation sequencing approach using an Ion Torrent PGM instrument. Primers for 15 target genes (NF1, RET, VHL, SDHA, SDHB, SDHC, SDHD, SDHAF2, TMEM127, MAX, MEN1, KIF1Bβ, EPAS1, CDKN2 & PHD2) were designed using ion ampliseq designer. Ion Reporter software and Ingenuity® Variant Analysis™ software (www.ingenuity.com/variants) from Ingenuity Systems were used to analysis these results. Overall, 713 variants were identified. The variants identified from the Ion Reporter ranged from 64 to 161 per patient. Single nucleotide variants (SNV) were the most common. Further annotation with the help of Ingenuity variant analysis revealed 29 of these 713variants were deletions. Of these, six variants were non-pathogenic and four were likely to be pathogenic. The remaining 19 variants were of uncertain significance. The most frequently altered gene in the cohort was KIF1B followed by NF1. Novel KIF1B pathogenic variant c.3375+1G>A was identified. The mutation was noted in a patient with clinically confirmed neurofibromatosis. Chromosome 1 showed the presence of maximum number of variants. Use of targeted next-generation sequencing is a sensitive method for the detecting genetic changes in patients with phaeochromocytoma/paraganglioma. The precise detection of these genetic changes helps in understanding the pathogenesis of these tumours. Copyright © 2016 Elsevier Inc. All rights reserved.

  11. Systems Biology-Based Investigation of Cellular Antiviral Drug Targets Identified by Gene-Trap Insertional Mutagenesis

    Science.gov (United States)

    Zhao, Junfei; Sheng, Jinsong; Rubin, Donald H.

    2016-01-01

    Viruses require host cellular factors for successful replication. A comprehensive systems-level investigation of the virus-host interactome is critical for understanding the roles of host factors with the end goal of discovering new druggable antiviral targets. Gene-trap insertional mutagenesis is a high-throughput forward genetics approach to randomly disrupt (trap) host genes and discover host genes that are essential for viral replication, but not for host cell survival. In this study, we used libraries of randomly mutagenized cells to discover cellular genes that are essential for the replication of 10 distinct cytotoxic mammalian viruses, 1 gram-negative bacterium, and 5 toxins. We herein reported 712 candidate cellular genes, characterizing distinct topological network and evolutionary signatures, and occupying central hubs in the human interactome. Cell cycle phase-specific network analysis showed that host cell cycle programs played critical roles during viral replication (e.g. MYC and TAF4 regulating G0/1 phase). Moreover, the viral perturbation of host cellular networks reflected disease etiology in that host genes (e.g. CTCF, RHOA, and CDKN1B) identified were frequently essential and significantly associated with Mendelian and orphan diseases, or somatic mutations in cancer. Computational drug repositioning framework via incorporating drug-gene signatures from the Connectivity Map into the virus-host interactome identified 110 putative druggable antiviral targets and prioritized several existing drugs (e.g. ajmaline) that may be potential for antiviral indication (e.g. anti-Ebola). In summary, this work provides a powerful methodology with a tight integration of gene-trap insertional mutagenesis testing and systems biology to identify new antiviral targets and drugs for the development of broadly acting and targeted clinical antiviral therapeutics. PMID:27632082

  12. A combined approach to data mining of textual and structured data to identify cancer-related targets

    Directory of Open Access Journals (Sweden)

    Adelstein S James

    2006-07-01

    Full Text Available Abstract Background We present an effective, rapid, systematic data mining approach for identifying genes or proteins related to a particular interest. A selected combination of programs exploring PubMed abstracts, universal gene/protein databases (UniProt, InterPro, NCBI Entrez, and state-of-the-art pathway knowledge bases (LSGraph and Ingenuity Pathway Analysis was assembled to distinguish enzymes with hydrolytic activities that are expressed in the extracellular space of cancer cells. Proteins were identified with respect to six types of cancer occurring in the prostate, breast, lung, colon, ovary, and pancreas. Results The data mining method identified previously undetected targets. Our combined strategy applied to each cancer type identified a minimum of 375 proteins expressed within the extracellular space and/or attached to the plasma membrane. The method led to the recognition of human cancer-related hydrolases (on average, ~35 per cancer type, among which were prostatic acid phosphatase, prostate-specific antigen, and sulfatase 1. Conclusion The combined data mining of several databases overcame many of the limitations of querying a single database and enabled the facile identification of gene products. In the case of cancer-related targets, it produced a list of putative extracellular, hydrolytic enzymes that merit additional study as candidates for cancer radioimaging and radiotherapy. The proposed data mining strategy is of a general nature and can be applied to other biological databases for understanding biological functions and diseases.

  13. Novel in vivo targets of ΔNp63 in keratinocytes identified by a modified chromatin immunoprecipitation approach

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    Ortt Kori

    2007-05-01

    Full Text Available Abstract Background p63 is a transcription factor that plays an important role in skin epidermal development and differentiation. The p63 gene encodes for two major protein isoforms, those containing an amino-terminal trans-activation domain (TAp63 and those lacking this domain (ΔNp63. Both the TA and ΔN transcripts are also alternatively spliced at the 3' end producing proteins with unique C-termini that are designated as α, β and γ isoforms. Recent research has suggested that ΔNp63 is the predominant isoform expressed and active in keratinocytes. Results To better elucidate the biological role of p63 in regulating gene expression in keratinocytes we performed chromatin immunoprecipitation (ChIP experiments with ΔNp63-specific antibodies. We included an additional step in the ChIP procedure to enrich for ΔNp63 targets by screening the library of immunoprecipitated DNA for its ability to bind recombinant GST-ΔNp63. Cloning of ΔNp63-ChIP-derived DNA fragments identified more than 60 potential ΔNp63 target loci that were located close to or embedded within known or predicted genes. Identity of these target genes suggests that they may participate in a myriad of cellular processes including transcriptional regulation, signaling and metabolism. Here we confirm the binding of ΔNp63 to several of these genomic loci both by EMSA and replicate ChIP assays. Finally we show that the expression of many of these target genes is altered when ΔNp63 levels in keratinocytes are reduced by siRNA, further confirming that these are bona fide targets. Conclusion This unbiased genomic approach has allowed us to uncover functional targets of ΔNp63 and serves as the initial step in further analysis of the transcriptional regulatory mechanisms that are governed by p63 in keratinocytes.

  14. Differential Gene Expression in the Meristem and during Early Fruit Growth of Pisum sativum L. Identifies Potential Targets for Breeding

    Science.gov (United States)

    Smitha Ninan, Annu; Shah, Anish; Song, Jiancheng; Jameson, Paula E.

    2017-01-01

    For successful molecular breeding it is important to identify targets to the gene family level, and in the specific species of interest, in this case Pisum sativum L. The cytokinins have been identified as a key breeding target due to their influence on plant architecture, and on seed size and sink activity. We focused on the cytokinin biosynthetic gene family (the IPTs) and the gene family key to the destruction of cytokinins (the CKXs), as well as other gene families potentially affected by changing cytokinin levels. These included key meristem genes (WUS and BAM1) and the transporter gene families, sucrose transporters (SUTs) and amino acid permeases (AAPs). We used reverse transcription quantitative PCR (RT-qPCR) to monitor gene expression in the vegetative meristem and in pre- and post-fertilisation young pea fruits. PsWUS expression was specific to the shoot apical meristem while PsBAM1 was highly expressed in the shoot apical meristem (SAM) but was also expressed at a low level in the young fruit. Differential expression was shown between genes and within gene families for IPT, CKX, SUT, and AAP. PsCKX7 showed strong gene family member-specific expression in the SAM, and was also expressed in young pea fruits. We suggest that PsCKX7 is a potential target for downregulation via molecular breeding or gene editing. PMID:28212324

  15. Differential Gene Expression in the Meristem and during Early Fruit Growth of Pisum sativum L. Identifies Potential Targets for Breeding

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    Annu Smitha Ninan

    2017-02-01

    Full Text Available For successful molecular breeding it is important to identify targets to the gene family level, and in the specific species of interest, in this case Pisum sativum L. The cytokinins have been identified as a key breeding target due to their influence on plant architecture, and on seed size and sink activity. We focused on the cytokinin biosynthetic gene family (the IPTs and the gene family key to the destruction of cytokinins (the CKXs, as well as other gene families potentially affected by changing cytokinin levels. These included key meristem genes (WUS and BAM1 and the transporter gene families, sucrose transporters (SUTs and amino acid permeases (AAPs. We used reverse transcription quantitative PCR (RT-qPCR to monitor gene expression in the vegetative meristem and in pre- and post-fertilisation young pea fruits. PsWUS expression was specific to the shoot apical meristem while PsBAM1 was highly expressed in the shoot apical meristem (SAM but was also expressed at a low level in the young fruit. Differential expression was shown between genes and within gene families for IPT, CKX, SUT, and AAP. PsCKX7 showed strong gene family member-specific expression in the SAM, and was also expressed in young pea fruits. We suggest that PsCKX7 is a potential target for downregulation via molecular breeding or gene editing.

  16. Protein-protein interaction networks identify targets which rescue the MPP+ cellular model of Parkinson’s disease

    Science.gov (United States)

    Keane, Harriet; Ryan, Brent J.; Jackson, Brendan; Whitmore, Alan; Wade-Martins, Richard

    2015-11-01

    Neurodegenerative diseases are complex multifactorial disorders characterised by the interplay of many dysregulated physiological processes. As an exemplar, Parkinson’s disease (PD) involves multiple perturbed cellular functions, including mitochondrial dysfunction and autophagic dysregulation in preferentially-sensitive dopamine neurons, a selective pathophysiology recapitulated in vitro using the neurotoxin MPP+. Here we explore a network science approach for the selection of therapeutic protein targets in the cellular MPP+ model. We hypothesised that analysis of protein-protein interaction networks modelling MPP+ toxicity could identify proteins critical for mediating MPP+ toxicity. Analysis of protein-protein interaction networks constructed to model the interplay of mitochondrial dysfunction and autophagic dysregulation (key aspects of MPP+ toxicity) enabled us to identify four proteins predicted to be key for MPP+ toxicity (P62, GABARAP, GBRL1 and GBRL2). Combined, but not individual, knockdown of these proteins increased cellular susceptibility to MPP+ toxicity. Conversely, combined, but not individual, over-expression of the network targets provided rescue of MPP+ toxicity associated with the formation of autophagosome-like structures. We also found that modulation of two distinct proteins in the protein-protein interaction network was necessary and sufficient to mitigate neurotoxicity. Together, these findings validate our network science approach to multi-target identification in complex neurological diseases.

  17. Differential Gene Expression in the Meristem and during Early Fruit Growth of Pisum sativum L. Identifies Potential Targets for Breeding.

    Science.gov (United States)

    Smitha Ninan, Annu; Shah, Anish; Song, Jiancheng; Jameson, Paula E

    2017-02-16

    For successful molecular breeding it is important to identify targets to the gene family level, and in the specific species of interest, in this case Pisum sativum L. The cytokinins have been identified as a key breeding target due to their influence on plant architecture, and on seed size and sink activity. We focused on the cytokinin biosynthetic gene family (the IPTs) and the gene family key to the destruction of cytokinins (the CKXs), as well as other gene families potentially affected by changing cytokinin levels. These included key meristem genes (WUS and BAM1) and the transporter gene families, sucrose transporters (SUTs) and amino acid permeases (AAPs). We used reverse transcription quantitative PCR (RT-qPCR) to monitor gene expression in the vegetative meristem and in pre- and post-fertilisation young pea fruits. PsWUS expression was specific to the shoot apical meristem while PsBAM1 was highly expressed in the shoot apical meristem (SAM) but was also expressed at a low level in the young fruit. Differential expression was shown between genes and within gene families for IPT, CKX, SUT, and AAP. PsCKX7 showed strong gene family member-specific expression in the SAM, and was also expressed in young pea fruits. We suggest that PsCKX7 is a potential target for downregulation via molecular breeding or gene editing.

  18. Malaria in pregnancy; facts from the parasitology laboratory: a ten-year study in Abuja, North Central Nigeria

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    Ibecheozor, N.K.O

    2014-09-01

    Full Text Available Malaria, which is transmitted by female Anopheles mosquitoes, is the major cause of mortality among the pregnant women in the sub-Saharan Africa. A ten year study of malaria in pregnancy was carried out in Abuja, North Central Nigeria. Thick and thin blood films were stained with the Giemsa methodology. Of the 16760 pregnant women blood samples, 4571 (27.3% were positive for malaria parasites caused by Plasmodium falciparum. Of the 4571 positive cases, 75 (1.7% had parasite density of >5000 parasites/µl of blood; 148 (3.2% had between 500-5000 parasites/µl of blood; 520 (11.4% had between 50 - 500 parasites/µl of blood; while 3828 (83.7% had between 5-50 parasites/µl of blood. With the current estimate of over 4500 deaths of pregnant women in Nigeria due to malaria annually, we must make deliberate efforts to stop these unacceptable and painful losses. The continued use of malaria rapid diagnostic tests (M-RDTs methodologies should be discontinued because of its negative implications. Therefore, the microscopic laboratory diagnostic component should be included in ANC at all level of health care facility.

  19. Detection of anti-Chlamydia trachomatis Antibodies in Patients with Acquired Immune Deficiency Syndrome in Abuja, Nigeria

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    Izebe, K. S.

    2008-01-01

    Full Text Available Chlamydia trachomatis (CT infections are among the sexually transmitted diseases known to increase the risk for human immunodeficiency virus infection. Serum samples from 34 consenting AIDS patients which attended the Government-approved Antiretroviral Treatment (ART Facility at the National Institute for Pharmaceutical Research and Development (NIPRD, Abuja between April 2005 and March 2006 were screened by enzyme immunoassay (EIA for the presence of anti-CT antibodies using ImmunoComb® Chlamydia Bivalent IgG Test kit (Orgenics, Israel. Anti-CT antibodies were detected in ten (29.4% of the thirty-four patients tested. The detection rate was higher among the females (33.3% than the males (23.1%. Patients of the age group 31-45 years had the highest detection of anti-C. trachomatis antibodies, followed by those of age group 16-30 years. The result of the present study suggests the presence of anti-CT antibodies in AIDS patients, and reinforces the need for routine screening for anti-CT antibodies as a necessary intervention to reduce the burden of chlamydial diseases and to reduce the risk of HIV and its spread in Nigeria. The outcome of this study also provides justification for the possible inclusion of anti-chlamydial agents in the National AIDS Management Plan to treat associated C. trachomatis infections.

  20. A new screening method to identify inhibitors of the Lol (localization of lipoproteins) system, a novel antibacterial target.

    Science.gov (United States)

    Ito, Hideaki; Ura, Atsushi; Oyamada, Yoshihiro; Yoshida, Hiroaki; Yamagishi, Jun-Ichi; Narita, Shin-Ichiro; Matsuyama, Shin-Ichi; Tokuda, Hajime

    2007-01-01

    As the Lol system, which is involved in localization of lipoproteins, is essential for Escherichia coli growth and widely conserved among gram-negative bacteria, it is considered to be a promising target for the development of anti-gram-negative bacterial agents. However, no high-throughput screening method has so far been developed to screen for Lol system inhibitors. By combining three assay systems (anucleate cell blue assay, Lpp assay, and LolA-dependent release inhibition assay) and a drug susceptibility test, we have successfully developed a new screening method for identification of compounds that inhibit the Lol system. Using this new screening method, we screened 23,600 in-house chemical compounds and found 2 Lol system inhibitors. We therefore conclude that our new screening method can efficiently identify new antibacterial agents that target the Lol system.

  1. Identifying mRNA targets of microRNA dysregulated in cancer: with application to clear cell Renal Cell Carcinoma

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    Liou Louis S

    2010-04-01

    Full Text Available Abstract Background MicroRNA regulate mRNA levels in a tissue specific way, either by inducing degradation of the transcript or by inhibiting translation or transcription. Putative mRNA targets of microRNA identified from seed sequence matches are available in many databases. However, such matches have a high false positive rate and cannot identify tissue specificity of regulation. Results We describe a simple method to identify direct mRNA targets of microRNA dysregulated in cancers from expression level measurements in patient matched tumor/normal samples. The word "direct" is used here in a strict sense to: a represent mRNA which have an exact seed sequence match to the microRNA in their 3'UTR, b the seed sequence match is strictly conserved across mouse, human, rat and dog genomes, c the mRNA and microRNA expression levels can distinguish tumor from normal with high significance and d the microRNA/mRNA expression levels are strongly and significantly anti-correlated in tumor and/or normal samples. We apply and validate the method using clear cell Renal Cell Carcinoma (ccRCC and matched normal kidney samples, limiting our analysis to mRNA targets which undergo degradation of the mRNA transcript because of a perfect seed sequence match. Dysregulated microRNA and mRNA are first identified by comparing their expression levels in tumor vs normal samples. Putative dysregulated microRNA/mRNA pairs are identified from these using seed sequence matches, requiring that the seed sequence be conserved in human/dog/rat/mouse genomes. These are further pruned by requiring a strong anti-correlation signature in tumor and/or normal samples. The method revealed many new regulations in ccRCC. For instance, loss of miR-149, miR-200c and mir-141 causes gain of function of oncogenes (KCNMA1, LOX, VEGFA and SEMA6A respectively and increased levels of miR-142-3p, miR-185, mir-34a, miR-224, miR-21 cause loss of function of tumor suppressors LRRC2, PTPN13, SFRP1

  2. An Integrative Transcriptomic Analysis for Identifying Novel Target Genes Corresponding to Severity Spectrum in Spinal Muscular Atrophy.

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    Chung-Wei Yang

    Full Text Available Spinal muscular atrophy (SMA is an inherited neuromuscular disease resulting from a recessive mutation in the SMN1 gene. This disease affects multiple organ systems with varying degrees of severity. Exploration of the molecular pathological changes occurring in different cell types in SMA is crucial for developing new therapies. This study collected 39 human microarray datasets from ArrayExpress and GEO databases to build an integrative transcriptomic analysis for recognizing novel SMA targets. The transcriptomic analysis was conducted through combining weighted correlation network analysis (WGCNA for gene module detection, gene set enrichment analysis (GSEA for functional categorization and filtration, and Cytoscape (visual interaction gene network analysis for target gene identification. Seven novel target genes (Bmp4, Serpine1, Gata6, Ptgs2, Bcl2, IL6 and Cntn1 of SMA were revealed, and are all known in the regulation of TNFα for controlling neural, cardiac and bone development. Sequentially, the differentially expressed patterns of these 7 target genes in mouse tissues (e.g., spinal cord, heart, muscles and bone were validated in SMA mice of different severities (pre-symptomatic, mildly symptomatic, and severely symptomatic. In severely symptomatic SMA mice, TNFα was up-regulated with attenuation of Bmp4 and increase of Serpine1 and Gata6 (a pathway in neural and cardiac development, but not in pre-symptomatic and mildly symptomatic SMA mice. The severely symptomatic SMA mice also had the elevated levels of Ptgs2 and Bcl2 (a pathway in skeletal development as well as IL6 and Cntn1 (a pathway in nervous system development. Thus, the 7 genes identified in this study might serve as potential target genes for future investigations of disease pathogenesis and SMA therapy.

  3. Global tyrosine kinome profiling of human thyroid tumors identifies Src as a promising target for invasive cancers

    Energy Technology Data Exchange (ETDEWEB)

    Cho, Nancy L., E-mail: nlcho@partners.org [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Lin, Chi-Iou [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Du, Jinyan [Broad Institute, Massachusetts Institute of Technology, Cambridge, MA 02142 (United States); Whang, Edward E. [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States); Ito, Hiromichi [Department of Surgery, Michigan State University, Lansing, MI 48912 (United States); Moore, Francis D.; Ruan, Daniel T. [Department of Surgery, Brigham and Women' s Hospital, Boston, MA 02115 (United States)

    2012-05-11

    Highlights: Black-Right-Pointing-Pointer Kinome profiling is a novel technique for identifying activated kinases in human cancers. Black-Right-Pointing-Pointer Src activity is increased in invasive thyroid cancers. Black-Right-Pointing-Pointer Inhibition of Src activity decreased proliferation and invasion in vitro. Black-Right-Pointing-Pointer Further investigation of Src targeted therapies in thyroid cancer is warranted. -- Abstract: Background: Novel therapies are needed for the treatment of invasive thyroid cancers. Aberrant activation of tyrosine kinases plays an important role in thyroid oncogenesis. Because current targeted therapies are biased toward a small subset of tyrosine kinases, we conducted a study to reveal novel therapeutic targets for thyroid cancer using a bead-based, high-throughput system. Methods: Thyroid tumors and matched normal tissues were harvested from twenty-six patients in the operating room. Protein lysates were analyzed using the Luminex immunosandwich, a bead-based kinase phosphorylation assay. Data was analyzed using GenePattern 3.0 software and clustered according to histology, demographic factors, and tumor status regarding capsular invasion, size, lymphovascular invasion, and extrathyroidal extension. Survival and invasion assays were performed to determine the effect of Src inhibition in papillary thyroid cancer (PTC) cells. Results: Tyrosine kinome profiling demonstrated upregulation of nine tyrosine kinases in tumors relative to matched normal thyroid tissue: EGFR, PTK6, BTK, HCK, ABL1, TNK1, GRB2, ERK, and SRC. Supervised clustering of well-differentiated tumors by histology, gender, age, or size did not reveal significant differences in tyrosine kinase activity. However, supervised clustering by the presence of invasive disease showed increased Src activity in invasive tumors relative to non-invasive tumors (60% v. 0%, p < 0.05). In vitro, we found that Src inhibition in PTC cells decreased cell invasion and proliferation

  4. A miRNA signature of chemoresistant mesenchymal phenotype identifies novel molecular targets associated with advanced pancreatic cancer.

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    Alakesh Bera

    Full Text Available In this study a microRNA (miRNA signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC cell line model (BxPC3-GZR and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13. Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.

  5. A miRNA signature of chemoresistant mesenchymal phenotype identifies novel molecular targets associated with advanced pancreatic cancer.

    Science.gov (United States)

    Bera, Alakesh; VenkataSubbaRao, Kolaparthi; Manoharan, Muthu Saravanan; Hill, Ping; Freeman, James W

    2014-01-01

    In this study a microRNA (miRNA) signature was identified in a gemcitabine resistant pancreatic ductal adenocarcinoma (PDAC) cell line model (BxPC3-GZR) and this signature was further examined in advanced PDAC tumor specimens from The Cancer Genome Atlas (TCGA) database. BxPC3-GZR showed a mesenchymal phenotype, expressed high levels of CD44 and showed a highly significant deregulation of 17 miRNAs. Based on relevance to cancer, a seven-miRNA signature (miR-100, miR-125b, miR-155, miR-21, miR-205, miR-27b and miR-455-3p) was selected for further studies. A strong correlation was observed for six of the seven miRNAs in 43 advanced tumor specimens compared to normal pancreas tissue. To assess the functional relevance we initially focused on miRNA-125b, which is over-expressed in both the BxPC3-GZR model and advanced PDAC tumor specimens. Knockdown of miRNA-125b in BxPC3-GZR and Panc-1 cells caused a partial reversal of the mesenchymal phenotype and enhanced response to gemcitabine. Moreover, RNA-seq data from each of 40 advanced PDAC tumor specimens from the TCGA data base indicate a negative correlation between expression of miRNA-125b and five of six potential target genes (BAP1, BBC3, NEU1, BCL2, STARD13). Thus far, two of these target genes, BBC3 and NEU1, that are tumor suppressor genes but not yet studied in PDAC, appear to be functional targets of miR-125b since knockdown of miR125b caused their up regulation. These miRNAs and their molecular targets may serve as targets to enhance sensitivity to chemotherapy and reduce metastatic spread.

  6. Landscape of Targeted Anti-Cancer Drug Synergies in Melanoma Identifies a Novel BRAF-VEGFR/PDGFR Combination Treatment.

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    Adam A Friedman

    Full Text Available A newer generation of anti-cancer drugs targeting underlying somatic genetic driver events have resulted in high single-agent or single-pathway response rates in selected patients, but few patients achieve complete responses and a sizeable fraction of patients relapse within a year. Thus, there is a pressing need for identification of combinations of targeted agents which induce more complete responses and prevent disease progression. We describe the results of a combination screen of an unprecedented scale in mammalian cells performed using a collection of targeted, clinically tractable agents across a large panel of melanoma cell lines. We find that even the most synergistic drug pairs are effective only in a discrete number of cell lines, underlying a strong context dependency for synergy, with strong, widespread synergies often corresponding to non-specific or off-target drug effects such as multidrug resistance protein 1 (MDR1 transporter inhibition. We identified drugs sensitizing cell lines that are BRAFV600E mutant but intrinsically resistant to BRAF inhibitor PLX4720, including the vascular endothelial growth factor receptor/kinase insert domain receptor (VEGFR/KDR and platelet derived growth factor receptor (PDGFR family inhibitor cediranib. The combination of cediranib and PLX4720 induced apoptosis in vitro and tumor regression in animal models. This synergistic interaction is likely due to engagement of multiple receptor tyrosine kinases (RTKs, demonstrating the potential of drug- rather than gene-specific combination discovery approaches. Patients with elevated biopsy KDR expression showed decreased progression free survival in trials of mitogen-activated protein kinase (MAPK kinase pathway inhibitors. Thus, high-throughput unbiased screening of targeted drug combinations, with appropriate library selection and mechanistic follow-up, can yield clinically-actionable drug combinations.

  7. Identifying the Types of Ion Channel-Targeted Conotoxins by Incorporating New Properties of Residues into Pseudo Amino Acid Composition

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    Yun Wu

    2016-01-01

    Full Text Available Conotoxins are a kind of neurotoxin which can specifically interact with potassium, sodium type, and calcium channels. They have become potential drug candidates to treat diseases such as chronic pain, epilepsy, and cardiovascular diseases. Thus, correctly identifying the types of ion channel-targeted conotoxins will provide important clue to understand their function and find potential drugs. Based on this consideration, we developed a new computational method to rapidly and accurately predict the types of ion-targeted conotoxins. Three kinds of new properties of residues were proposed to use in pseudo amino acid composition to formulate conotoxins samples. The support vector machine was utilized as classifier. A feature selection technique based on F-score was used to optimize features. Jackknife cross-validated results showed that the overall accuracy of 94.6% was achieved, which is higher than other published results, demonstrating that the proposed method is superior to published methods. Hence the current method may play a complementary role to other existing methods for recognizing the types of ion-target conotoxins.

  8. Identifying Molecular Targets for New Drug Development for Chronic Obstructive Pulmonary Disease: What Does the Future Hold?

    Science.gov (United States)

    Barnes, Peter J

    2015-08-01

    There is an urgent need to develop more effective therapies for chronic obstructive pulmonary disease (COPD) that target the underlying inflammatory disease process. Current therapies with long-acting bronchodilators and inhaled corticosteroids fail to prevent either disease progression or mortality, as they do not suppress the underlying inflammation. With better understanding of the inflammatory and destructive process in the pathophysiology of COPD, several new therapeutic targets have been identified. Several mediator antagonists or inhibitors tested in COPD have so far been disappointing. Broad-spectrum anti-inflammatory drugs may be more effective, and include inhibitors of the proinflammatory enzymes phosphodiesterase-4, p38 mitogen-activated protein kinase, Janus-activated kinases, NF-κB kinase, and PI3kinase-γ and -δ, but side effects after oral administration are a major limitation; therefore, in future inhaled delivery may be necessary. A new promising approach is reversal of corticosteroid resistance through increasing histone deacetylase-2 activity. This might be achieved by existing treatments such as theophylline, nortriptyline, and macrolides, or more selectively by PI3kinase-δ inhibitors. Other treatments in development target oxidative stress, the failure to resolve inflammation, aberrant repair mechanisms, and accelerated lung aging.

  9. Acetylome study in mouse adipocytes identifies targets of SIRT1 deacetylation in chromatin organization and RNA processing.

    Science.gov (United States)

    Kim, Sun-Yee; Sim, Choon Kiat; Tang, Hui; Han, Weiping; Zhang, Kangling; Xu, Feng

    2016-05-15

    SIRT1 is a key protein deacetylase that regulates cellular metabolism through lysine deacetylation on both histones and non-histone proteins. Lysine acetylation is a wide-spread post-translational modification found on many regulatory proteins and it plays an essential role in cell signaling, transcription and metabolism. In mice, SIRT1 has known protective functions during high-fat diet but the acetylome regulated by SIRT1 in adipocytes is not completely understood. Here we conducted acetylome analyses in murine adipocytes treated with small-molecule modulators that inhibit or activate the deacetylase activity of SIRT1. We identified a total of 302 acetylated peptides from 78 proteins in this study. From the list of potential SIRT1 targets, we selected seven candidates and further verified that six of them can be deacetylated by SIRT1 in-vitro. Among them, half of the SIRT1 targets are involved in regulating chromatin structure and the other half is involved in RNA processing. Our results provide a resource for further SIRT1 target validation in fat cells and suggest a potential role of SIRT1 in the regulation of chromatin structure and RNA processing, which may possibly extend to other cell types as well.

  10. Network modeling identifies molecular functions targeted by miR-204 to suppress head and neck tumor metastasis.

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    Younghee Lee

    2010-04-01

    Full Text Available Due to the large number of putative microRNA gene targets predicted by sequence-alignment databases and the relative low accuracy of such predictions which are conducted independently of biological context by design, systematic experimental identification and validation of every functional microRNA target is currently challenging. Consequently, biological studies have yet to identify, on a genome scale, key regulatory networks perturbed by altered microRNA functions in the context of cancer. In this report, we demonstrate for the first time how phenotypic knowledge of inheritable cancer traits and of risk factor loci can be utilized jointly with gene expression analysis to efficiently prioritize deregulated microRNAs for biological characterization. Using this approach we characterize miR-204 as a tumor suppressor microRNA and uncover previously unknown connections between microRNA regulation, network topology, and expression dynamics. Specifically, we validate 18 gene targets of miR-204 that show elevated mRNA expression and are enriched in biological processes associated with tumor progression in squamous cell carcinoma of the head and neck (HNSCC. We further demonstrate the enrichment of bottleneckness, a key molecular network topology, among miR-204 gene targets. Restoration of miR-204 function in HNSCC cell lines inhibits the expression of its functionally related gene targets, leads to the reduced adhesion, migration and invasion in vitro and attenuates experimental lung metastasis in vivo. As importantly, our investigation also provides experimental evidence linking the function of microRNAs that are located in the cancer-associated genomic regions (CAGRs to the observed predisposition to human cancers. Specifically, we show miR-204 may serve as a tumor suppressor gene at the 9q21.1-22.3 CAGR locus, a well established risk factor locus in head and neck cancers for which tumor suppressor genes have not been identified. This new strategy

  11. Topological analysis of protein co-abundance networks identifies novel host targets important for HCV infection and pathogenesis

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    McDermott Jason E

    2012-04-01

    Full Text Available Abstract Background High-throughput methods for obtaining global measurements of transcript and protein levels in biological samples has provided a large amount of data for identification of 'target' genes and proteins of interest. These targets may be mediators of functional processes involved in disease and therefore represent key points of control for viruses and bacterial pathogens. Genes and proteins that are the most highly differentially regulated are generally considered to be the most important. We present topological analysis of co-abundance networks as an alternative to differential regulation for confident identification of target proteins from two related global proteomics studies of hepatitis C virus (HCV infection. Results We analyzed global proteomics data sets from a cell culture study of HCV infection and from a clinical study of liver biopsies from HCV-positive patients. Using lists of proteins known to be interaction partners with pathogen proteins we show that the most differentially regulated proteins in both data sets are indeed enriched in pathogen interactors. We then use these data sets to generate co-abundance networks that link proteins based on similar abundance patterns in time or across patients. Analysis of these co-abundance networks using a variety of network topology measures revealed that both degree and betweenness could be used to identify pathogen interactors with better accuracy than differential regulation alone, though betweenness provides the best discrimination. We found that though overall differential regulation was not correlated between the cell culture and liver biopsy data, network topology was conserved to an extent. Finally, we identified a set of proteins that has high betweenness topology in both networks including a protein that we have recently shown to be essential for HCV replication in cell culture. Conclusions The results presented show that the network topology of protein co

  12. Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing.

    Science.gov (United States)

    Faucon, Frederic; Dusfour, Isabelle; Gaude, Thierry; Navratil, Vincent; Boyer, Frederic; Chandre, Fabrice; Sirisopa, Patcharawan; Thanispong, Kanutcharee; Juntarajumnong, Waraporn; Poupardin, Rodolphe; Chareonviriyaphap, Theeraphap; Girod, Romain; Corbel, Vincent; Reynaud, Stephane; David, Jean-Philippe

    2015-09-01

    The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations.

  13. Three-step HPLC-ESI-MS/MS procedure for screening and identifying non-target flavonoid derivatives

    Science.gov (United States)

    Rak, Gábor; Fodor, Péter; Abrankó, László

    2010-02-01

    A three-step HPLC-ESI-MS/MS procedure is designed for screening and identification of non-target flavonoid derivatives of selected flavonoid aglycones. In this method the five commonly appearing aglycones (apigenin, luteolin, myricetin, naringenin and quercetin) were selected. The method consists of three individual mass spectrometric experiments of which the first two were implemented within a single chromatographic acquisition. The third step was carried out during a replicate chromatographic run using the same RP-HPLC conditions. The first step, a multiple reaction monitoring (MRM) scan of the aglycones was performed to define the number of derivatives relating to the selected aglycones. For this purpose the characteristic aglycone parts of the unknowns were used as specific tags of the molecules, which were generated as in-source fragments. Secondly, a full scan MS experiment is performed to identify the masses of the potential derivatives of the selected aglycones. Finally, the third step had the capability to confirm the supposed derivatives. The developed method was applied to a commercially available black currant juice to demonstrate its capability to detect and identify various flavonoid glycosides without any preliminary information about their presence in the sample. As a result 13 compounds were detected and identified in total. Namely, 3 different myricetin glycosides and the myricetin aglycone 2 luteolin glycosides plus the aglycone and 3 quercetin glycosides plus the aglycone could be identified from the tested black currant sample. In the case of apigenin and naringenin only the aglycones could be detected.

  14. Identifying genomic changes associated with insecticide resistance in the dengue mosquito Aedes aegypti by deep targeted sequencing

    Science.gov (United States)

    Faucon, Frederic; Dusfour, Isabelle; Gaude, Thierry; Navratil, Vincent; Boyer, Frederic; Chandre, Fabrice; Sirisopa, Patcharawan; Thanispong, Kanutcharee; Juntarajumnong, Waraporn; Poupardin, Rodolphe; Chareonviriyaphap, Theeraphap; Girod, Romain; Corbel, Vincent; Reynaud, Stephane; David, Jean-Philippe

    2015-01-01

    The capacity of mosquitoes to resist insecticides threatens the control of diseases such as dengue and malaria. Until alternative control tools are implemented, characterizing resistance mechanisms is crucial for managing resistance in natural populations. Insecticide biodegradation by detoxification enzymes is a common resistance mechanism; however, the genomic changes underlying this mechanism have rarely been identified, precluding individual resistance genotyping. In particular, the role of copy number variations (CNVs) and polymorphisms of detoxification enzymes have never been investigated at the genome level, although they can represent robust markers of metabolic resistance. In this context, we combined target enrichment with high-throughput sequencing for conducting the first comprehensive screening of gene amplifications and polymorphisms associated with insecticide resistance in mosquitoes. More than 760 candidate genes were captured and deep sequenced in several populations of the dengue mosquito Ae. aegypti displaying distinct genetic backgrounds and contrasted resistance levels to the insecticide deltamethrin. CNV analysis identified 41 gene amplifications associated with resistance, most affecting cytochrome P450s overtranscribed in resistant populations. Polymorphism analysis detected more than 30,000 variants and strong selection footprints in specific genomic regions. Combining Bayesian and allele frequency filtering approaches identified 55 nonsynonymous variants strongly associated with resistance. Both CNVs and polymorphisms were conserved within regions but differed across continents, confirming that genomic changes underlying metabolic resistance to insecticides are not universal. By identifying novel DNA markers of insecticide resistance, this study opens the way for tracking down metabolic changes developed by mosquitoes to resist insecticides within and among populations. PMID:26206155

  15. Proteasome activator complex PA28 identified as an accessible target in prostate cancer by in vivo selection of human antibodies

    Science.gov (United States)

    Sánchez-Martín, David; Martínez-Torrecuadrada, Jorge; Teesalu, Tambet; Sugahara, Kazuki N.; Alvarez-Cienfuegos, Ana; Ximénez-Embún, Pilar; Fernández-Periáñez, Rodrigo; Martín, M. Teresa; Molina-Privado, Irene; Ruppen-Cañás, Isabel; Blanco-Toribio, Ana; Cañamero, Marta; Cuesta, Ángel M.; Compte, Marta; Kremer, Leonor; Bellas, Carmen; Alonso-Camino, Vanesa; Guijarro-Muñoz, Irene; Sanz, Laura; Ruoslahti, Erkki; Alvarez-Vallina, Luis

    2013-01-01

    Antibody cancer therapies rely on systemically accessible targets and suitable antibodies that exert a functional activity or deliver a payload to the tumor site. Here, we present proof-of-principle of in vivo selection of human antibodies in tumor-bearing mice that identified a tumor-specific antibody able to deliver a payload and unveils the target antigen. By using an ex vivo enrichment process against freshly disaggregated tumors to purge the repertoire, in combination with in vivo biopanning at optimized phage circulation time, we have identified a human domain antibody capable of mediating selective localization of phage to human prostate cancer xenografts. Affinity chromatography followed by mass spectrometry analysis showed that the antibody recognizes the proteasome activator complex PA28. The specificity of soluble antibody was confirmed by demonstrating its binding to the active human PA28αβ complex. Whereas systemically administered control phage was confined in the lumen of blood vessels of both normal tissues and tumors, the selected phage spread from tumor vessels into the perivascular tumor parenchyma. In these areas, the selected phage partially colocalized with PA28 complex. Furthermore, we found that the expression of the α subunit of PA28 [proteasome activator complex subunit 1 (PSME1)] is elevated in primary and metastatic human prostate cancer and used anti-PSME1 antibodies to show that PSME1 is an accessible marker in mouse xenograft tumors. These results support the use of PA28 as a tumor marker and a potential target for therapeutic intervention in prostate cancer. PMID:23918357

  16. A multi-element screening method to identify metal targets for blood biomonitoring in green sea turtles (Chelonia mydas).

    Science.gov (United States)

    Villa, C A; Finlayson, S; Limpus, C; Gaus, C

    2015-04-15

    Biomonitoring of blood is commonly used to identify and quantify occupational or environmental exposure to chemical contaminants. Increasingly, this technique has been applied to wildlife contaminant monitoring, including for green turtles, allowing for the non-lethal evaluation of chemical exposure in their nearshore environment. The sources, composition, bioavailability and toxicity of metals in the marine environment are, however, often unknown and influenced by numerous biotic and abiotic factors. These factors can vary considerably across time and space making the selection of the most informative elements for biomonitoring challenging. This study aimed to validate an ICP-MS multi-element screening method for green turtle blood in order to identify and facilitate prioritisation of target metals for subsequent fully quantitative analysis. Multi-element screening provided semiquantitative results for 70 elements, 28 of which were also determined through fully quantitative analysis. Of the 28 comparable elements, 23 of the semiquantitative results had an accuracy between 67% and 112% relative to the fully quantified values. In lieu of any available turtle certified reference materials (CRMs), we evaluated the use of human blood CRMs as a matrix surrogate for quality control, and compared two commonly used sample preparation methods for matrix related effects. The results demonstrate that human blood provides an appropriate matrix for use as a quality control material in the fully quantitative analysis of metals in turtle blood. An example for the application of this screening method is provided by comparing screening results from blood of green turtles foraging in an urban and rural region in Queensland, Australia. Potential targets for future metal biomonitoring in these regions were identified by this approach.

  17. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    Energy Technology Data Exchange (ETDEWEB)

    Sarró, Eduard, E-mail: eduard.sarro@vhir.org [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Jacobs-Cachá, Conxita, E-mail: conxita.jacobs@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Itarte, Emilio, E-mail: emili.itarte@uab.es [Departament de Bioquímica i Biologia Molecular, Unitat de Bioquímica de Biociències, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain); Meseguer, Anna, E-mail: ana.meseguer@vhir.org [Renal Physiopathology, CIBBIM-Nanomedicine, Vall d' Hebron Research Institute (VHIR), 08035 Barcelona (Spain); Departament de Bioquimica i Biologia Molecular, Facultat de Medicina, Universitat Autònoma de Barcelona, 08193 Bellaterra (Barcelona) (Spain)

    2012-01-15

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC. Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also identified ER

  18. Analyses of a Glycine max Degradome Library Identify microRNA Targets and MicroRNAs that Trigger Secondary SiRNA Biogenesis

    Institute of Scientific and Technical Information of China (English)

    Zheng Hu; Qiyan Jiang; Zhiyong Ni; Rui Chen; Shuo Xu; Hui Zhang

    2013-01-01

    Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs.In this study,a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced.428 potential targets of small interfering RNAs and 25 novel miRNA families were identified.A total of 211 potential miRNA targets,including 174 conserved miRNA targets and 37 soybeanspecific miRNA targets,were identified.Among them,121 targets were first discovered in soybean.The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing.The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean,and nine Auxin Response Factors were identified as TAS3 siRNA targets.Twentythree miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean.These targets were guided by five miRNAs:gma-miR393,gma-miR1508,gma-miR1510,gma-miR1514,and novel-11.Multiple targets of these secondary siRNAs were detected.These 23 miRNA targets may be the putative novel TAS genes in soybean.Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.

  19. Analyses of a Glycine max degradome library identify microRNA targets and microRNAs that trigger secondary siRNA biogenesis.

    Science.gov (United States)

    Hu, Zheng; Jiang, Qiyan; Ni, Zhiyong; Chen, Rui; Xu, Shuo; Zhang, Hui

    2013-02-01

    Plant microRNAs (miRNAs) regulate gene expression mainly by guiding cleavage of target mRNAs. In this study, a degradome library constructed from different soybean (Glycine max (L.) Merr.) tissues was deep-sequenced. 428 potential targets of small interfering RNAs and 25 novel miRNA families were identified. A total of 211 potential miRNA targets, including 174 conserved miRNA targets and 37 soybean-specific miRNA targets, were identified. Among them, 121 targets were first discovered in soybean. The signature distribution of soybean primary miRNAs (pri-miRNAs) showed that most pri-miRNAs had the characteristic pattern of Dicer processing. The biogenesis of TAS3 small interfering RNAs (siRNAs) was conserved in soybean, and nine Auxin Response Factors were identified as TAS3 siRNA targets. Twenty-three miRNA targets produced secondary small interfering RNAs (siRNAs) in soybean. These targets were guided by five miRNAs: gma-miR393, gma-miR1508, gma-miR1510, gma-miR1514, and novel-11. Multiple targets of these secondary siRNAs were detected. These 23 miRNA targets may be the putative novel TAS genes in soybean. Global identification of miRNA targets and potential novel TAS genes will contribute to research on the functions of miRNAs in soybean.

  20. Evaluation of soil corrosivity and aquifer protective capacity using geoelectrical investigation in Bwari basement complex area, Abuja

    Indian Academy of Sciences (India)

    A E Adeniji; O V Omonona; D N Obiora; J U Chukudebelu

    2014-04-01

    Bwari is one of the six municipal area councils of the Federal Capital Territory (FCT), Abuja with its attendant growing population and infrastructural developments. Groundwater is the main source of water supply in the area, and urbanization and industrialization are the predominant contributors of contaminants to the hydrological systems. In order to guarantee a continuous supply of potable water, there is a need to investigate the vulnerability of the aquifers to contaminants emanating from domestic and industrial wastes. A total of 20 vertical electrical soundings using Schlumberger electrode array with a maximum half current electrodes separation of 300 m was employed. The results show that the area is characterized by 3–6 geoelectric subsurface layers. The measured overburden thickness ranges from 1.0 to 24.3 m, with a mean value of 7.4 m. The resistivity and longitudinal conductance of the overburden units range from 18 to 11,908 m and 0.047 to 0.875 mhos, respectively. Areas considered as high corrosivity are the central parts with > 180 m. The characteristic longitudinal unit conductance was used to classify the area into zones of good (0.7–4.49 mhos), moderate (0.2–0.69 mhos), weak (0.1–0.19 mhos), and poor (> 0.1) aquifer protective capacity. Zones characterized by materials of moderate to good protective capacity serve as sealing potential for the underlying hydrogeological system in the area. This study is aimed at delineating zones that are very prone to groundwater contamination from surface contaminants and subsurface soils that are corrosive to utility pipes buried underground. Hence the findings of this work will constitute part of the tools for groundwater development and management and structural/infrastructural development planning of the area.

  1. Utilisation of the National Antiretroviral Therapy Guidelines among health care professionals working in Abuja treatment centres, Nigeria

    Directory of Open Access Journals (Sweden)

    Lindiwe I. Zungu

    2009-04-01

    Full Text Available Background: Access to and utilisations of the National Antiretroviral Treatment Guidelines (NATG are valuable factors for effective programme implementation. The objective of this study was to investigate the accessibility of the NATG and their utilisation by health care professionals from five treatment centres in Abuja, Nigeria.Method: A quantitative cross-sectional descriptive survey was conducted in 2007 using purposively sampled health care professionals. Questionnaires were self-administered to participants who consented in writing to participate in the survey.Results: 97 health care professionals participated in this study with about equal numbers of men and women: 48 (49.5% women and 49 (50.5% men. Of these, 21.6% were unaware of the existence of the NATG in their treatment centres. More than half (51.5% reported that they did not have access to the NATG as opposed to those (48.5% who had access to the guidelines. Furthermore, 16.5% of the participants confirmed that they had access to an institutional copy of the NATG while 14.4% indicated that they had individual copies and only 3.1% stated that they had individual copies and access to the hospital copy as well. Regarding utilisation of the NATG, 41.2% rarely used them, 32.9% never used them and only 25.7% often used them. The most frequent use of the NATG was among pharmacists (38.1% compared to the least frequent use among nurses (20.0%.Conclusion: Poor accessibility of the NATG may have a negative impact on guidelines utilisation among health care professionals in Nigeria.

  2. FUNCTIONAL GENOMICS IDENTIFIES TIS21-DEPENDENT MECHANISMS AND PUTATIVE CANCER DRUG TARGETS UNDERLYING MEDULLOBLASTOMA SHH-TYPE DEVELOPMENT

    Directory of Open Access Journals (Sweden)

    Giulia Gentile

    2016-11-01

    Full Text Available We have recently generated a novel medulloblastoma (MB mouse model with activation of the Shh pathway and lacking the MB suppressor Tis21 (Patched1+-Tis21KO.ts main phenotype is a defect of migration of the cerebellar granule precursor cells (GCPs. By genomic analysis of GCPs in vivo, we identified as drug target and major responsible of this defect the down-regulation of the promigratory chemokine Cxcl3. Consequently, the GCPs remain longer in the cerebellum proliferative area, and the MB frequency is enhanced. Here, we further analyzed the genes deregulated in a Tis21-dependent manner (Patched1+-is21 wild-type versus Ptch1+-Tis21 knockout, among which are a number of down-regulated tumor inhibitors and up-regulated tumor facilitators, focusing on pathways potentially involved in the tumorigenesis and on putative new drug targets.The data analysis using bioinformatic tools revealed: i a link between the Shh signaling and the Tis21-dependent impairment of the GCPs migration, through a Shh-dependent deregulation of the clathrin-mediated chemotaxis operating in the primary cilium through the Cxcl3-Cxcr2 axis; ii a possible lineage shift of Shh-type GCPs toward retinal precursor phenotype the neural cell type involved in group 3 MB; iii the identification of a subset of putative drug targets for MB, involved, among the others, in the regulation of Hippo signaling and centrosome assembly. Finally, our findings define also the role of Tis21 in the regulation of gene expression, through epigenetic and RNA processing mechanisms, influencing the fate of the GCPs.

  3. Early T Cell Recognition of B Cells following Epstein-Barr Virus Infection: Identifying Potential Targets for Prophylactic Vaccination.

    Directory of Open Access Journals (Sweden)

    Jill M Brooks

    2016-04-01

    Full Text Available Epstein-Barr virus, a B-lymphotropic herpesvirus, is the cause of infectious mononucleosis, has strong aetiologic links with several malignancies and has been implicated in certain autoimmune diseases. Efforts to develop a prophylactic vaccine to prevent or reduce EBV-associated disease have, to date, focused on the induction of neutralising antibody responses. However, such vaccines might be further improved by inducing T cell responses capable of recognising and killing recently-infected B cells. In that context, EBNA2, EBNA-LP and BHRF1 are the first viral antigens expressed during the initial stage of B cell growth transformation, yet have been poorly characterised as CD8+ T cell targets. Here we describe CD8+ T cell responses against each of these three "first wave" proteins, identifying target epitopes and HLA restricting alleles. While EBNA-LP and BHRF1 each contained one strong CD8 epitope, epitopes within EBNA2 induced immunodominant responses through several less common HLA class I alleles (e.g. B*3801 and B*5501, as well as subdominant responses through common class I alleles (e.g. B7 and C*0304. Importantly, such EBNA2-specific CD8+ T cells recognised B cells within the first day post-infection, prior to CD8+ T cells against well-characterised latent target antigens such as EBNA3B or LMP2, and effectively inhibited outgrowth of EBV-transformed B cell lines. We infer that "first wave" antigens of the growth-transforming infection, especially EBNA2, constitute potential CD8+ T cell immunogens for inclusion in prophylactic EBV vaccine design.

  4. Large Dog Relinquishment to Two Municipal Facilities in New York City and Washington, D.C.: Identifying Targets for Intervention

    Directory of Open Access Journals (Sweden)

    Emily Weiss

    2014-07-01

    Full Text Available While the overall trend in euthanasia has been decreasing nationally, large dogs are at a higher risk of euthanasia than other sized dogs in most animal shelters in the United States. We hypothesized one way to increase the lives saved with respect to these large dogs is to keep them home when possible. In order to develop solutions to decrease relinquishment, a survey was developed to learn more about the reasons owners relinquish large dogs. The survey was administered to owners relinquishing their dogs at two large municipal facilities, one in New York City and one in Washington, D.C. There were 157 responses between the two facilities. We found both significant similarities and differences between respondents and their dogs from the two cities. We identified opportunities to potentially support future relinquishers and found that targets for interventions are likely different in each community.

  5. Developing clinical practice guidelines: target audiences, identifying topics for guidelines, guideline group composition and functioning and conflicts of interest

    Directory of Open Access Journals (Sweden)

    Eccles Martin P

    2012-07-01

    Full Text Available Abstract Clinical practice guidelines are one of the foundations of efforts to improve health care. In 1999, we authored a paper about methods to develop guidelines. Since it was published, the methods of guideline development have progressed both in terms of methods and necessary procedures and the context for guideline development has changed with the emergence of guideline clearing houses and large scale guideline production organisations (such as the UK National Institute for Health and Clinical Excellence. It therefore seems timely to, in a series of three articles, update and extend our earlier paper. In this first paper we discuss: the target audience(s for guidelines and their use of guidelines; identifying topics for guidelines; guideline group composition (including consumer involvement and the processes by which guideline groups function and the important procedural issue of managing conflicts of interest in guideline development.

  6. Developing clinical practice guidelines: target audiences, identifying topics for guidelines, guideline group composition and functioning and conflicts of interest.

    Science.gov (United States)

    Eccles, Martin P; Grimshaw, Jeremy M; Shekelle, Paul; Schünemann, Holger J; Woolf, Steven

    2012-07-04

    Clinical practice guidelines are one of the foundations of efforts to improve health care. In 1999, we authored a paper about methods to develop guidelines. Since it was published, the methods of guideline development have progressed both in terms of methods and necessary procedures and the context for guideline development has changed with the emergence of guideline clearing houses and large scale guideline production organisations (such as the UK National Institute for Health and Clinical Excellence). It therefore seems timely to, in a series of three articles, update and extend our earlier paper. In this first paper we discuss: the target audience(s) for guidelines and their use of guidelines; identifying topics for guidelines; guideline group composition (including consumer involvement) and the processes by which guideline groups function and the important procedural issue of managing conflicts of interest in guideline development.

  7. Genome-Wide Association Study with Targeted and Non-targeted NMR Metabolomics Identifies 15 Novel Loci of Urinary Human Metabolic Individuality.

    Directory of Open Access Journals (Sweden)

    Johannes Raffler

    2015-09-01

    Full Text Available Genome-wide association studies with metabolic traits (mGWAS uncovered many genetic variants that influence human metabolism. These genetically influenced metabotypes (GIMs contribute to our metabolic individuality, our capacity to respond to environmental challenges, and our susceptibility to specific diseases. While metabolic homeostasis in blood is a well investigated topic in large mGWAS with over 150 known loci, metabolic detoxification through urinary excretion has only been addressed by few small mGWAS with only 11 associated loci so far. Here we report the largest mGWAS to date, combining targeted and non-targeted 1H NMR analysis of urine samples from 3,861 participants of the SHIP-0 cohort and 1,691 subjects of the KORA F4 cohort. We identified and replicated 22 loci with significant associations with urinary traits, 15 of which are new (HIBCH, CPS1, AGXT, XYLB, TKT, ETNPPL, SLC6A19, DMGDH, SLC36A2, GLDC, SLC6A13, ACSM3, SLC5A11, PNMT, SLC13A3. Two-thirds of the urinary loci also have a metabolite association in blood. For all but one of the 6 loci where significant associations target the same metabolite in blood and urine, the genetic effects have the same direction in both fluids. In contrast, for the SLC5A11 locus, we found increased levels of myo-inositol in urine whereas mGWAS in blood reported decreased levels for the same genetic variant. This might indicate less effective re-absorption of myo-inositol in the kidneys of carriers. In summary, our study more than doubles the number of known loci that influence urinary phenotypes. It thus allows novel insights into the relationship between blood homeostasis and its regulation through excretion. The newly discovered loci also include variants previously linked to chronic kidney disease (CPS1, SLC6A13, pulmonary hypertension (CPS1, and ischemic stroke (XYLB. By establishing connections from gene to disease via metabolic traits our results provide novel hypotheses about molecular

  8. A statistically inferred microRNA network identifies breast cancer target miR-940 as an actin cytoskeleton regulator

    Science.gov (United States)

    Bhajun, Ricky; Guyon, Laurent; Pitaval, Amandine; Sulpice, Eric; Combe, Stéphanie; Obeid, Patricia; Haguet, Vincent; Ghorbel, Itebeddine; Lajaunie, Christian; Gidrol, Xavier

    2015-02-01

    MiRNAs are key regulators of gene expression. By binding to many genes, they create a complex network of gene co-regulation. Here, using a network-based approach, we identified miRNA hub groups by their close connections and common targets. In one cluster containing three miRNAs, miR-612, miR-661 and miR-940, the annotated functions of the co-regulated genes suggested a role in small GTPase signalling. Although the three members of this cluster targeted the same subset of predicted genes, we showed that their overexpression impacted cell fates differently. miR-661 demonstrated enhanced phosphorylation of myosin II and an increase in cell invasion, indicating a possible oncogenic miRNA. On the contrary, miR-612 and miR-940 inhibit phosphorylation of myosin II and cell invasion. Finally, expression profiling in human breast tissues showed that miR-940 was consistently downregulated in breast cancer tissues

  9. A structure-based strategy to identify new molecular scaffolds targeting the bacterial ribosomal A-site.

    Science.gov (United States)

    Foloppe, Nicolas; Chen, I-Jen; Davis, Ben; Hold, Adam; Morley, Dave; Howes, Rob

    2004-03-01

    The need for novel antibiotics is widely recognized. A well validated target of antibiotics is the bacterial ribosome. Recent X-ray structures of the ribosome bound to antibiotics have shed new light on the binding sites of these antibiotics, providing fresh impetus for structure-based strategies aiming at identifying new ribosomal ligands. In that respect, the ribosomal decoding region of the aminoacyl-tRNA acceptor site (A-site) is of particular interest because oligonucleotide model systems of this site are available for crystallography, NMR and compound binding assays. This work presents how these different resources can be combined in a hierarchical screening strategy which has led to the identification of new A-site ligands. The approach exploits an X-ray structure of the A-site against which large and diverse libraries of compounds were computationally docked. The complementarity of the compounds to the A-site was assessed using a scoring function specifically calibrated for RNA targets. Starting from approximately 1 million compounds, the computational selection of candidate ligands allowed us to focus the experimental work on 129 compounds, 34 of which showed affinity for the A-site in a FRET-based binding assay. NMR experiments confirmed binding to the A-site for some compounds. For the most potent compound in the FRET assay, a tentative binding mode is suggested, which is compatible with the NMR data and the limited SAR in this series. Overall, the results validate the screening strategy.

  10. Targeting of MAPK-associated molecules identifies SON as a prime target to attenuate the proliferation and tumorigenicity of pancreatic cancer cells

    Directory of Open Access Journals (Sweden)

    Furukawa Toru

    2012-12-01

    Full Text Available Abstract Background Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinase (MAPK. Activation of MAPK is associated with the upregulation of genes implicated in the proliferation and survival of pancreatic cancer cells. We hypothesized that knockdown of these MAPK-associated molecules could produce notable anticancer phenotypes. Methods A RNA interference-mediated knockdown screening of 78 MAPK-associated molecules previously identified was performed to find molecules specifically associated with proliferation of pancreatic cancer cells in vitro. Expression of an identified molecule in pancreatic cancer tissues was examined by immunohistochemistry. In vivo tumorigenicity of cancer cells with stable knockdown of the molecule was assayed by using xenograft models. Flow cytometry and live cell imaging were employed to assess an association of the molecule with cell cycle. Results The knockdown screening revealed that knockdown of SON, the gene encoding SON, which is a large serine/arginine-rich protein involved in RNA processing, substantially suppressed pancreatic cancer cell proliferation and survival in vitro and tumorigenicity in vivo. SON expression was higher in ductal adenocarcinomas than in cells of normal ducts and precursor lesions in pancreatic cancer tissues. Knockdown of SON induced G2/M arrest and apoptosis in cultured cancer cells. The suppressive effect of SON knockdown on proliferation was less pronounced in cultured normal duct epithelial cells. SON formed nuclear speckles in the interphase of the cell cycle and dispersed in the cytoplasm during mitosis. Live cell imaging showed that SON diffusely dispersed in the early mitotic phase, accumulated in some foci in the cytoplasm in the late mitotic phase, and gradually reassembled into speckles after mitosis. Conclusion These results indicate that SON plays a critical role in the proliferation, survival, and tumorigenicity of pancreatic

  11. A high throughput screen identifies Nefopam as targeting cell proliferation in β-catenin driven neoplastic and reactive fibroproliferative disorders.

    Directory of Open Access Journals (Sweden)

    Raymond Poon

    Full Text Available Fibroproliferative disorders include neoplastic and reactive processes (e.g. desmoid tumor and hypertrophic scars. They are characterized by activation of β-catenin signaling, and effective pharmacologic approaches are lacking. Here we undertook a high throughput screen using human desmoid tumor cell cultures to identify agents that would inhibit cell viability in tumor cells but not normal fibroblasts. Agents were then tested in additional cell cultures for an effect on cell proliferation, apoptosis, and β-catenin protein level. Ultimately they were tested in Apc1638N mice, which develop desmoid tumors, as well as in wild type mice subjected to full thickness skin wounds. The screen identified Neofopam, as an agent that inhibited cell numbers to 42% of baseline in cell cultures from β-catenin driven fibroproliferative disorders. Nefopam decreased cell proliferation and β-catenin protein level to 50% of baseline in these same cell cultures. The half maximal effective concentration in-vitro was 0.5 uM and there was a plateau in the effect after 48 hours of treatment. Nefopam caused a 45% decline in tumor number, 33% decline in tumor volume, and a 40% decline in scar size when tested in mice. There was also a 50% decline in β-catenin level in-vivo. Nefopam targets β-catenin protein level in mesenchymal cells in-vitro and in-vivo, and may be an effective therapy for neoplastic and reactive processes driven by β-catenin mediated signaling.

  12. Cancer in silico drug discovery: a systems biology tool for identifying candidate drugs to target specific molecular tumor subtypes.

    Science.gov (United States)

    San Lucas, F Anthony; Fowler, Jerry; Chang, Kyle; Kopetz, Scott; Vilar, Eduardo; Scheet, Paul

    2014-12-01

    Large-scale cancer datasets such as The Cancer Genome Atlas (TCGA) allow researchers to profile tumors based on a wide range of clinical and molecular characteristics. Subsequently, TCGA-derived gene expression profiles can be analyzed with the Connectivity Map (CMap) to find candidate drugs to target tumors with specific clinical phenotypes or molecular characteristics. This represents a powerful computational approach for candidate drug identification, but due to the complexity of TCGA and technology differences between CMap and TCGA experiments, such analyses are challenging to conduct and reproduce. We present Cancer in silico Drug Discovery (CiDD; scheet.org/software), a computational drug discovery platform that addresses these challenges. CiDD integrates data from TCGA, CMap, and Cancer Cell Line Encyclopedia (CCLE) to perform computational drug discovery experiments, generating hypotheses for the following three general problems: (i) determining whether specific clinical phenotypes or molecular characteristics are associated with unique gene expression signatures; (ii) finding candidate drugs to repress these expression signatures; and (iii) identifying cell lines that resemble the tumors being studied for subsequent in vitro experiments. The primary input to CiDD is a clinical or molecular characteristic. The output is a biologically annotated list of candidate drugs and a list of cell lines for in vitro experimentation. We applied CiDD to identify candidate drugs to treat colorectal cancers harboring mutations in BRAF. CiDD identified EGFR and proteasome inhibitors, while proposing five cell lines for in vitro testing. CiDD facilitates phenotype-driven, systematic drug discovery based on clinical and molecular data from TCGA.

  13. Combining in vitro protein detection and in vivo antibody detection identifies potential vaccine targets against Staphylococcus aureus during osteomyelitis.

    Science.gov (United States)

    den Reijer, P Martijn; Sandker, Marjan; Snijders, Susan V; Tavakol, Mehri; Hendrickx, Antoni P A; van Wamel, Willem J B

    2017-02-01

    Currently, little is known about the in vivo human immune response against Staphylococcus aureus during a biofilm-associated infection, such as osteomyelitis, and how this relates to protein production in biofilms in vitro. Therefore, we characterized IgG responses in 10 patients with chronic osteomyelitis against 50 proteins of S. aureus, analyzed the presence of these proteins in biofilms of the infecting isolates on polystyrene (PS) and human bone in vitro, and explored the relation between in vivo and in vitro data. IgG levels against 15 different proteins were significantly increased in patients compared to healthy controls. Using a novel competitive Luminex-based assay, eight of these proteins [alpha toxin, Staphylococcus aureus formyl peptide receptor-like 1 inhibitor (FlipR), glucosaminidase, iron-responsive surface determinants A and H, the putative ABC transporter SACOL0688, staphylococcal complement inhibitor (SCIN), and serine-aspartate repeat-containing protein E (SdrE)] were also detected in a majority of the infecting isolates during biofilm formation in vitro. However, 4 other proteins were detected in only a minority of isolates in vitro while, vice versa, 7 proteins were detected in multiple isolates in vitro but not associated with significantly increased IgG levels in patients. Detection of proteins was largely confirmed using a transcriptomic approach. Our data provide further insights into potential therapeutic targets, such as for vaccination, to reduce S. aureus virulence and biofilm formation. At the same time, our data suggest that either in vitro or immunological in vivo data alone should be interpreted cautiously and that combined studies are necessary to identify potential targets.

  14. Intraoperative near-infrared fluorescence imaging targeting folate receptors identifies lung cancer in a large-animal model.

    Science.gov (United States)

    Keating, Jane J; Runge, Jeffrey J; Singhal, Sunil; Nims, Sarah; Venegas, Ollin; Durham, Amy C; Swain, Gary; Nie, Shuming; Low, Philip S; Holt, David E

    2017-05-15

    Complete tumor resection is the most important predictor of patient survival with non-small cell lung cancer. Methods for intraoperative margin assessment after lung cancer excision are lacking. This study evaluated near-infrared (NIR) intraoperative imaging with a folate-targeted molecular contrast agent (OTL0038) for the localization of primary lung adenocarcinomas, lymph node sampling, and margin assessment. Ten dogs with lung cancer underwent either video-assisted thoracoscopic surgery or open thoracotomy and tumor excision after an intravenous injection of OTL0038. Lungs were imaged with an NIR imaging device both in vivo and ex vivo. The wound bed was re-imaged for retained fluorescence suspicious for positive tumor margins. The tumor signal-to-background ratio (SBR) was measured in all cases. Next, 3 human patients were enrolled in a proof-of-principle study. Tumor fluorescence was measured both in situ and ex vivo. All canine tumors fluoresced in situ (mean Fluoptics SBR, 5.2 [range, 2.7-8.1]; mean Karl Storz SBR 1.9 [range, 1.4-2.6]). In addition, the fluorescence was consistent with tumor margins on pathology. Three positive lymph nodes were discovered with NIR imaging. Also, a positive retained tumor margin was discovered upon NIR imaging of the wound bed. Human pulmonary adenocarcinomas were also fluorescent both in situ and ex vivo (mean SBR, > 2.0). NIR imaging can identify lung cancer in a large-animal model. In addition, NIR imaging can discriminate lymph nodes harboring cancer cells and also bring attention to a positive tumor margin. In humans, pulmonary adenocarcinomas fluoresce after the injection of the targeted contrast agent. Cancer 2017;123:1051-60. © 2016 American Cancer Society. © 2016 American Cancer Society.

  15. Stimulation of oral fibroblast chemokine receptors identifies CCR3 and CCR4 as potential wound healing targets.

    Science.gov (United States)

    Buskermolen, Jeroen K; Roffel, Sanne; Gibbs, Susan

    2017-11-01

    The focus of this study was to determine which chemokine receptors are present on oral fibroblasts and whether these receptors influence proliferation, migration, and/or the release of wound healing mediators. This information may provide insight into the superior wound healing characteristics of the oral mucosa. The gingiva fibroblasts expressed 12 different chemokine receptors (CCR3, CCR4, CCR6, CCR9, CCR10, CXCR1, CXCR2, CXCR4, CXCR5, CXCR7, CX3CR1, and XCR1), as analyzed by flow cytometry. Fourteen corresponding chemokines (CCL5, CCL15, CCL20, CCL22, CCL25, CCL27, CCL28, CXCL1, CXCL8, CXCL11, CXCL12, CXCL13, CX3CL1, and XCL1) were used to study the activation of these receptors on gingiva fibroblasts. Twelve of these fourteen chemokines stimulated gingiva fibroblast migration (all except for CXCL8 and CXCL12). Five of the chemokines stimulated proliferation (CCL5/CCR3, CCL15/CCR3, CCL22/CCR4, CCL28/CCR3/CCR10, and XCL1/XCR1). Furthermore, CCL28/CCR3/CCR10 and CCL22/CCR4 stimulation increased IL-6 secretion and CCL28/CCR3/CCR10 together with CCL27/CCR10 upregulated HGF secretion. Moreover, TIMP-1 secretion was reduced by CCL15/CCR3. In conclusion, this in-vitro study identifies chemokine receptor-ligand pairs which may be used in future targeted wound healing strategies. In particular, we identified the chemokine receptors CCR3 and CCR4, and the mucosa specific chemokine CCL28, as having an predominant role in oral wound healing by increasing human gingiva fibroblast proliferation, migration, and the secretion of IL-6 and HGF and reducing the secretion of TIMP-1. © 2017 The Authors. Journal of Cellular Physiology Published by wiley periodicals, Inc.

  16. TARGET:?

    National Research Council Canada - National Science Library

    James M Acton

    2014-01-01

      By 2003. as military planners had become worried that the country's long-range conventional weapons, such as cruise missiles, might be too slow to reach hypothetical distant targets that needed to be struck urgently...

  17. Identifying new lignin bioengineering targets: impact of epicatechin, quercetin glycoside, and gallate derivatives on the lignification and fermentation of maize cell walls

    Science.gov (United States)

    Apoplastic targeting of secondary metabolites compatible with monolignol polymerization may provide new avenues for designing lignins that are less inhibitory toward fiber fermentation. To identify suitable monolignol substitutes, we artificially lignified maize cell walls with normal monolignols pl...

  18. Using environmental public health tracking to identify community targets for public health actions in childhood lead poisoning in Wisconsin.

    Science.gov (United States)

    Berney, Dawn; Camponeschi, Jenny; Coons, Marjorie; Creswell, Paul D; Schirmer, Joe; Walsh, Reghan

    2015-01-01

    In an effort to improve the ability of local public health departments to target resources to the highest need regions, the Wisconsin Environmental Public Health Tracking (WI EPHT) Program worked to enhance its public portal to benefit the Wisconsin Childhood Lead Poisoning Prevention Program (WCLPPP) and other programs. The WI EPHT Program conducted this enhancement in collaboration with WCLPPP. The WI EPHT enhanced public portal is the next phase of Wisconsin's ongoing efforts in environmental public health tracking. As part of this process, this new mapping application includes mapping capacity that provides information on childhood lead testing and results at county and census tract levels in Wisconsin. The WI EPHT Program will update its public portal to have the capability to map data at a subcounty level (ie, census tract or zip code) for some data topics when such data are available. This tool is available to local public health departments and other public health organizations throughout Wisconsin as a resource to identify communities most affected by the Centers for Disease Control and Prevention's new guidelines with regard to childhood lead poisoning. The collaboration between WI EPHT and WCLPPP on updating and enhancing the portal exemplifies the power of environmental health data to inform a more accurate understanding of public health problems.

  19. Chemical Genetics Identify eIF2α Kinase Heme Regulated Inhibitor as Anti-Cancer Target

    Science.gov (United States)

    Chen, Ting; Ozel, Duygu; Qiao, Yuan; Harbinski, Fred; Chen, Limo; Denoyelle, Séverine; He, Xiaoying; Zvereva, Nela; Supko, Jeffrey G.; Chorev, Michael; Halperin, Jose A.; Aktas, Bertal H.

    2013-01-01

    Translation initiation plays a critical role in cellular homeostasis, proliferation, differentiation and malignant transformation. Consistently, increasing the abundance of the eIF2·GTP·Met-tRNAi translation initiation complex transforms normal cells and contributes to cancer initiation and the severity of some anemia. The chemical modifiers of the eIF2·GTP·Met-tRNAi ternary complex are therefore invaluable tools for studying its role in the pathobiology of human disorders and for determining if this complex can be pharmacologically targeted for therapeutic purposes. Using a cell based assay, we identified N,N’-diarylureas as novel inhibitors of the ternary complex abundance. Direct functional-genetics and biochemical evidence demonstrated that the N,N’-diarylureas activate heme regulated inhibitor kinase, thereby phosphorylate eIF2α and reduce abundance of the ternary complex. Using tumor cell proliferation in vitro and tumor growth in vivo as paradigms, we demonstrate that N,N’-diarylureas are potent and specific tools for studying the role eIF2·GTP·Met-tRNAi ternary complex in the pathobiology of human disorders. PMID:21765405

  20. Network modelling reveals the mechanism underlying colitis-associated colon cancer and identifies novel combinatorial anti-cancer targets.

    Science.gov (United States)

    Lu, Junyan; Zeng, Hanlin; Liang, Zhongjie; Chen, Limin; Zhang, Liyi; Zhang, Hao; Liu, Hong; Jiang, Hualiang; Shen, Bairong; Huang, Ming; Geng, Meiyu; Spiegel, Sarah; Luo, Cheng

    2015-10-08

    The connection between inflammation and tumourigenesis has been well established. However, the detailed molecular mechanism underlying inflammation-associated tumourigenesis remains unknown because this process involves a complex interplay between immune microenvironments and epithelial cells. To obtain a more systematic understanding of inflammation-associated tumourigenesis as well as to identify novel therapeutic approaches, we constructed a knowledge-based network describing the development of colitis-associated colon cancer (CAC) by integrating the extracellular microenvironment and intracellular signalling pathways. Dynamic simulations of the CAC network revealed a core network module, including P53, MDM2, and AKT, that may govern the malignant transformation of colon epithelial cells in a pro-tumor inflammatory microenvironment. Furthermore, in silico mutation studies and experimental validations led to a novel finding that concurrently targeting ceramide and PI3K/AKT pathway by chemical probes or marketed drugs achieves synergistic anti-cancer effects. Overall, our network model can guide further mechanistic studies on CAC and provide new insights into the design of combinatorial cancer therapies in a rational manner.

  1. Transcriptome and proteome of Conus planorbis identify the nicotinic receptors as primary target for the defensive venom.

    Science.gov (United States)

    Jin, Ai-Hua; Vetter, Irina; Himaya, Siddhihalu W A; Alewood, Paul F; Lewis, Richard J; Dutertre, Sébastien

    2015-12-01

    Most venomous predators have evolved complex venom primarily to immobilize their prey and secondarily to defend against predators. In a new paradigm, carnivorous marine gastropods of the genus Conus were shown to rapidly and reversibly switch between two types of venoms in response to predatory or defensive stimulus, suggesting that the defensive use of venom may have a more important role in venom evolution and specialization than previously thought. To further investigate this phenomenon, the defensive repertoire of a vermivorous species, Conus planorbis, was deciphered using second-generation sequencing coupled to high-throughput proteomics. The venom gland transcriptome of C. planorbis revealed 182 unique conotoxin precursors from 25 gene superfamilies, with superfamily T dominating in terms of read and paralog numbers. Analysis of the defense-evoked venom revealed that this vermivorous species uses a similarly complex arsenal to deter aggressors as more recently evolved fish- and mollusk-hunting species, with MS/MS validating 23 conotoxin sequences from six superfamilies. Pharmacological characterization of the defensive venom on human receptors identified the nicotinic acetylcholine receptors as a primary target. This work provides the first insights into the composition and biological activity of specifically evolved defensive venoms in vermivorous cone snails. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  2. Antibody responses to NY-ESO-1 in primary breast cancer identify a subtype target for immunotherapy.

    Science.gov (United States)

    Hamaï, Ahmed; Duperrier-Amouriaux, Karine; Pignon, Pascale; Raimbaud, Isabelle; Memeo, Lorenzo; Colarossi, Cristina; Canzonieri, Vincenzo; Perin, Tiziana; Classe, Jean-Marc; Campone, Mario; Jézéquel, Pascal; Campion, Loïc; Ayyoub, Maha; Valmori, Danila

    2011-01-01

    The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited common characteristics, being mainly hormone receptor (HR)⁻ invasive ductal carcinomas of high grade, including both HER2⁻ and HER2⁺ tumors. In line with these results, we detected ESO expression in 20% of primary HR⁻ BC, including both ESO Ab⁺ and Ab⁻ patients, but not in HR⁺ BC. Interestingly, whereas expression levels in ESO⁺ BC were not significantly different between ESO Ab⁺ and Ab⁻ patients, the former had, in average, significantly higher numbers of tumor-infiltrated lymph nodes, indicating that lymph node invasion may be required for the development of spontaneous anti-tumor immune responses. Thus, the presence of ESO Ab identifies a tumor subtype of HR⁻ (HER2⁻ or HER2⁺) primary BC with frequent ESO expression and, together with the assessment of antigen expression in the tumor, may be instrumental for the selection of patients for whom ESO-based immunotherapy may complement standard therapy.

  3. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer*

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S.; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-01-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. PMID:26330541

  4. "By slapping their laps, the patient will know that you truly care for her": A qualitative study on social norms and acceptability of the mistreatment of women during childbirth in Abuja, Nigeria.

    Science.gov (United States)

    Bohren, Meghan A; Vogel, Joshua P; Tunçalp, Özge; Fawole, Bukola; Titiloye, Musibau A; Olutayo, Akinpelu Olanrewaju; Oyeniran, Agnes A; Ogunlade, Modupe; Metiboba, Loveth; Osunsan, Olubunmi R; Idris, Hadiza A; Alu, Francis E; Oladapo, Olufemi T; Gülmezoglu, A Metin; Hindin, Michelle J

    2016-12-01

    Many women experience mistreatment during childbirth in health facilities across the world. However, limited evidence exists on how social norms and attitudes of both women and providers influence mistreatment during childbirth. Contextually-specific evidence is needed to understand how normative factors affect how women are treated. This paper explores the acceptability of four scenarios of mistreatment during childbirth. Two facilities were identified in Abuja, Nigeria. Qualitative methods (in-depth interviews (IDIs) and focus group discussions (FGDs)) were used with a purposive sample of women, midwives, doctors and administrators. Participants were presented with four scenarios of mistreatment during childbirth: slapping, verbal abuse, refusing to help the woman and physical restraint. Thematic analysis was used to synthesize findings, which were interpreted within the study context and an existing typology of mistreatment during childbirth. Eighty-four IDIs and 4 FGDs are included in this analysis. Participants reported witnessing and experiencing mistreatment during childbirth, including slapping, physical restraint to a delivery bed, shouting, intimidation, and threats of physical abuse or poor health outcomes. Some women and providers considered each of the four scenarios as mistreatment. Others viewed these scenarios as appropriate and acceptable measures to gain compliance from the woman and ensure a good outcome for the baby. Women and providers blamed a woman's "disobedience" and "uncooperativeness" during labor for her experience of mistreatment. Blaming women for mistreatment parallels the intimate partner violence literature, demonstrating how traditional practices and low status of women potentiate gender inequality. These findings can be used to facilitate dialogue in Nigeria by engaging stakeholders to discuss how to challenge these norms and hold providers accountable for their actions. Until women and their families are able to freely condemn

  5. Targeted deep resequencing identifies coding variants in the PEAR1 gene that play a role in platelet aggregation.

    Directory of Open Access Journals (Sweden)

    Yoonhee Kim

    Full Text Available Platelet aggregation is heritable, and genome-wide association studies have detected strong associations with a common intronic variant of the platelet endothelial aggregation receptor1 (PEAR1 gene both in African American and European American individuals. In this study, we used a sequencing approach to identify additional exonic variants in PEAR1 that may also determine variability in platelet aggregation in the GeneSTAR Study. A 0.3 Mb targeted region on chromosome 1q23.1 including the entire PEAR1 gene was Sanger sequenced in 104 subjects (45% male, 49% African American, age = 52±13 selected on the basis of hyper- and hypo- aggregation across three different agonists (collagen, epinephrine, and adenosine diphosphate. Single-variant and multi-variant burden tests for association were performed. Of the 235 variants identified through sequencing, 61 were novel, and three of these were missense variants. More rare variants (MAF<5% were noted in African Americans compared to European Americans (108 vs. 45. The common intronic GWAS-identified variant (rs12041331 demonstrated the most significant association signal in African Americans (p = 4.020×10(-4; no association was seen for additional exonic variants in this group. In contrast, multi-variant burden tests indicated that exonic variants play a more significant role in European Americans (p = 0.0099 for the collective coding variants compared to p = 0.0565 for intronic variant rs12041331. Imputation of the individual exonic variants in the rest of the GeneSTAR European American cohort (N = 1,965 supports the results noted in the sequenced discovery sample: p = 3.56×10(-4, 2.27×10(-7, 5.20×10(-5 for coding synonymous variant rs56260937 and collagen, epinephrine and adenosine diphosphate induced platelet aggregation, respectively. Sequencing approaches confirm that a common intronic variant has the strongest association with platelet aggregation in African Americans

  6. A scalable method for molecular network reconstruction identifies properties of targets and mutations in acute myeloid leukemia.

    Science.gov (United States)

    Ong, Edison; Szedlak, Anthony; Kang, Yunyi; Smith, Peyton; Smith, Nicholas; McBride, Madison; Finlay, Darren; Vuori, Kristiina; Mason, James; Ball, Edward D; Piermarocchi, Carlo; Paternostro, Giovanni

    2015-04-01

    A key aim of systems biology is the reconstruction of molecular networks. We do not yet, however, have networks that integrate information from all datasets available for a particular clinical condition. This is in part due to the limited scalability, in terms of required computational time and power, of existing algorithms. Network reconstruction methods should also be scalable in the sense of allowing scientists from different backgrounds to efficiently integrate additional data. We present a network model of acute myeloid leukemia (AML). In the current version (AML 2.1), we have used gene expression data (both microarray and RNA-seq) from 5 different studies comprising a total of 771 AML samples and a protein-protein interactions dataset. Our scalable network reconstruction method is in part based on the well-known property of gene expression correlation among interacting molecules. The difficulty of distinguishing between direct and indirect interactions is addressed by optimizing the coefficient of variation of gene expression, using a validated gold-standard dataset of direct interactions. Computational time is much reduced compared to other network reconstruction methods. A key feature is the study of the reproducibility of interactions found in independent clinical datasets. An analysis of the most significant clusters, and of the network properties (intraset efficiency, degree, betweenness centrality, and PageRank) of common AML mutations demonstrated the biological significance of the network. A statistical analysis of the response of blast cells from 11 AML patients to a library of kinase inhibitors provided an experimental validation of the network. A combination of network and experimental data identified CDK1, CDK2, CDK4, and CDK6 and other kinases as potential therapeutic targets in AML.

  7. Phosphoproteomic Analysis Identifies Focal Adhesion Kinase 2 (FAK2) as a Potential Therapeutic Target for Tamoxifen Resistance in Breast Cancer.

    Science.gov (United States)

    Wu, Xinyan; Zahari, Muhammad Saddiq; Renuse, Santosh; Nirujogi, Raja Sekhar; Kim, Min-Sik; Manda, Srikanth S; Stearns, Vered; Gabrielson, Edward; Sukumar, Saraswati; Pandey, Akhilesh

    2015-11-01

    Tamoxifen, an estrogen receptor-α (ER) antagonist, is an important agent for the treatment of breast cancer. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying this resistance, we treated the MCF7 breast cancer cell line with tamoxifen for over six months and showed that this cell line acquired resistance to tamoxifen in vitro and in vivo. We performed SILAC-based quantitative phosphoproteomic profiling on the tamoxifen resistant and vehicle-treated sensitive cell lines to quantify the phosphorylation alterations associated with tamoxifen resistance. From >5600 unique phosphopeptides identified, 1529 peptides exhibited hyperphosphorylation and 409 peptides showed hypophosphorylation in the tamoxifen resistant cells. Gene set enrichment analysis revealed that focal adhesion pathway was one of the most enriched signaling pathways activated in tamoxifen resistant cells. Significantly, we showed that the focal adhesion kinase FAK2 was not only hyperphosphorylated but also transcriptionally up-regulated in tamoxifen resistant cells. FAK2 suppression by specific siRNA knockdown or a small molecule inhibitor repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 is significantly associated with shorter metastasis-free survival in estrogen receptor-positive breast cancer patients treated with tamoxifen. Our studies suggest that FAK2 is a potential therapeutic target for the management of hormone-refractory breast cancers. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris.

    Science.gov (United States)

    Luyet, Camille; Schulze, Katja; Sayar, Beyza S; Howald, Denise; Müller, Eliane J; Galichet, Arnaud

    2015-01-01

    The majority of pemphigus vulgaris (PV) patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis). The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg) 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG), PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice) as well as PV patients' biopsies (n=6). A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP) and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other diseases including

  9. Preclinical studies identify non-apoptotic low-level caspase-3 as therapeutic target in pemphigus vulgaris.

    Directory of Open Access Journals (Sweden)

    Camille Luyet

    Full Text Available The majority of pemphigus vulgaris (PV patients suffer from a live-threatening loss of intercellular adhesion between keratinocytes (acantholysis. The disease is caused by auto-antibodies that bind to desmosomal cadherins desmoglein (Dsg 3 or Dsg3 and Dsg1 in mucous membranes and skin. A currently unresolved controversy in PV is whether apoptosis is involved in the pathogenic process. The objective of this study was to perform preclinical studies to investigate apoptotic pathway activation in PV pathogenesis with the goal to assess its potential for clinical therapy. For this purpose, we investigated mouse and human skin keratinocyte cultures treated with PV antibodies (the experimental Dsg3 monospecific antibody AK23 or PV patients IgG, PV mouse models (passive transfer of AK23 or PVIgG into adult and neonatal mice as well as PV patients' biopsies (n=6. A combination of TUNEL assay, analyses of membrane integrity, early apoptotic markers such as cleaved poly-ADP-ribose polymerase (PARP and the collapse of actin cytoskeleton failed to provide evidence for apoptosis in PV pathogenesis. However, the in vitro and in vivo PV models, allowing to monitor progression of lesion formation, revealed an early, transient and low-level caspase-3 activation. Pharmacological inhibition confirmed the functional implication of caspase-3 in major events in PV such as shedding of Dsg3, keratin retraction, proliferation including c-Myc induction, p38MAPK activation and acantholysis. Together, these data identify low-level caspase-3 activation downstream of disrupted Dsg3 trans- or cis-adhesion as a major event in PV pathogenesis that is non-synonymous with apoptosis and represents, unlike apoptotic components, a promising target for clinical therapy. At a broader level, these results posit that an impairment of adhesive functions in concert with low-level, non-lethal caspase-3 activation can evoke profound cellular changes which may be of relevance for other

  10. Identifying policy target groups with qualitative and quantitative methods: the case of wildfire risk on nonindustrial private forest lands

    Science.gov (United States)

    A. Paige. Fischer

    2012-01-01

    Designing policies to harness the potential of heterogeneous target groups such as nonindustrial private forest owners to contribute to public policy goals can be challenging. The behaviors of such groups are shaped by their diverse motivations and circumstances. Segmenting heterogeneous target groups into more homogeneous subgroups may improve the chances of...

  11. Analysis of Settlement Expansion and Urban Growth Modelling Using Geoinformation for Assessing Potential Impacts of Urbanization on Climate in Abuja City, Nigeria

    Directory of Open Access Journals (Sweden)

    Mahmoud Ibrahim Mahmoud

    2016-03-01

    Full Text Available This study analyzed the spatiotemporal pattern of settlement expansion in Abuja, Nigeria, one of West Africa’s fastest developing cities, using geoinformation and ancillary datasets. Three epochs of Land-use Land-cover (LULC maps for 1986, 2001 and 2014 were derived from Landsat images using support vector machines (SVM. Accuracy assessment (AA of the LULC maps based on the pixel count resulted in overall accuracy of 82%, 92% and 92%, while the AA derived from the error adjusted area (EAA method stood at 69%, 91% and 91% for 1986, 2001 and 2014, respectively. Two major techniques for detecting changes in the LULC epochs involved the use of binary maps as well as a post-classification comparison approach. Quantitative spatiotemporal analysis was conducted to detect LULC changes with specific focus on the settlement development pattern of Abuja, the federal capital city (FCC of Nigeria. Logical transitions to the urban category were modelled for predicting future scenarios for the year 2050 using the embedded land change modeler (LCM in the IDRISI package. Based on the EAA, the result showed that urban areas increased by more than 11% between 1986 and 2001. In contrast, this value rose to 17% between 2001 and 2014. The LCM model projected LULC changes that showed a growing trend in settlement expansion, which might take over allotted spaces for green areas and agricultural land if stringent development policies and enforcement measures are not implemented. In conclusion, integrating geospatial technologies with ancillary datasets offered improved understanding of how urbanization processes such as increased imperviousness of such a magnitude could influence the urban microclimate through the alteration of natural land surface temperature. Urban expansion could also lead to increased surface runoff as well as changes in drainage geography leading to urban floods.

  12. Neurocognitive impairment associated with predominantly early stage HIV infection in Abuja, Nigeria.

    Science.gov (United States)

    Akolo, Christopher; Royal, Walter; Cherner, Mariana; Okwuasaba, Kanayo; Eyzaguirre, Lindsay; Adebiyi, Ruxton; Umlauf, Anya; Hendrix, Terence; Johnson, Joyce; Abimiku, Alashl'e; Blattner, William A

    2014-08-01

    assessments to identify areas of impairment that may be specifically linked to a history of HIV infection among individuals in Nigeria. Confirmation of these findings awaits analyses using data from a larger number of control subjects.

  13. Genomic approach to therapeutic target validation identifies a glucose-lowering GLP1R variant protective for coronary heart disease

    Science.gov (United States)

    Scott, Robert A.; Freitag, Daniel F.; Li, Li; Chu, Audrey Y.; Surendran, Praveen; Young, Robin; Grarup, Niels; Stancáková, Alena; Chen, Yuning; V.Varga, Tibor; Yaghootkar, Hanieh; Luan, Jian'an; Zhao, Jing Hua; Willems, Sara M.; Wessel, Jennifer; Wang, Shuai; Maruthur, Nisa; Michailidou, Kyriaki; Pirie, Ailith; van der Lee, Sven J.; Gillson, Christopher; Olama, Ali Amin Al; Amouyel, Philippe; Arriola, Larraitz; Arveiler, Dominique; Aviles-Olmos, Iciar; Balkau, Beverley; Barricarte, Aurelio; Barroso, Inês; Garcia, Sara Benlloch; Bis, Joshua C.; Blankenberg, Stefan; Boehnke, Michael; Boeing, Heiner; Boerwinkle, Eric; Borecki, Ingrid B.; Bork-Jensen, Jette; Bowden, Sarah; Caldas, Carlos; Caslake, Muriel; Cupples, L. Adrienne; Cruchaga, Carlos; Czajkowski, Jacek; den Hoed, Marcel; Dunn, Janet A.; Earl, Helena M.; Ehret, Georg B.; Ferrannini, Ele; Ferrieres, Jean; Foltynie, Thomas; Ford, Ian; Forouhi, Nita G.; Gianfagna, Francesco; Gonzalez, Carlos; Grioni, Sara; Hiller, Louise; Jansson, Jan-Håkan; Jørgensen, Marit E.; Jukema, J. Wouter; Kaaks, Rudolf; Kee, Frank; Kerrison, Nicola D.; Key, Timothy J.; Kontto, Jukka; Kote-Jarai, Zsofia; Kraja, Aldi T.; Kuulasmaa, Kari; Kuusisto, Johanna; Linneberg, Allan; Liu, Chunyu; Marenne, Gaëlle; Mohlke, Karen L.; Morris, Andrew P.; Muir, Kenneth; Müller-Nurasyid, Martina; Munroe, Patricia B.; Navarro, Carmen; Nielsen, Sune F.; Nilsson, Peter M.; Nordestgaard, Børge G.; Packard, Chris J.; Palli, Domenico; Panico, Salvatore; Peloso, Gina M.; Perola, Markus; Peters, Annette; Poole, Christopher J.; Quirós, J. Ramón; Rolandsson, Olov; Sacerdote, Carlotta; Salomaa, Veikko; Sánchez, María-José; Sattar, Naveed; Sharp, Stephen J.; Sims, Rebecca; Slimani, Nadia; Smith, Jennifer A.; Thompson, Deborah J.; Trompet, Stella; Tumino, Rosario; van der A, Daphne L.; van der Schouw, Yvonne T.; Virtamo, Jarmo; Walker, Mark; Walter, Klaudia; Abraham, Jean E.; Amundadottir, Laufey T.; Aponte, Jennifer L.; Butterworth, Adam S.; Dupuis, Josée; Easton, Douglas F.; Eeles, Rosalind A.; Erdmann, Jeanette; Franks, Paul W.; Frayling, Timothy M.; Hansen, Torben; Howson, Joanna M. M.; Jørgensen, Torben; Kooner, Jaspal; Laakso, Markku; Langenberg, Claudia; McCarthy, Mark I.; Pankow, James S.; Pedersen, Oluf; Riboli, Elio; Rotter, Jerome I.; Saleheen, Danish; Samani, Nilesh J.; Schunkert, Heribert; Vollenweider, Peter; O'Rahilly, Stephen; Deloukas, Panos; Danesh, John; Goodarzi, Mark O.; Kathiresan, Sekar; Meigs, James B.; Ehm, Margaret G.; Wareham, Nicholas J.; Waterworth, Dawn M.

    2016-01-01

    Regulatory authorities have indicated that new drugs to treat type 2 diabetes (T2D) should not be associated with an unacceptable increase in cardiovascular risk. Human genetics may be able to inform development of antidiabetic therapies by predicting cardiovascular and other health endpoints. We therefore investigated the association of variants in 6 genes that encode drug targets for obesity or T2D with a range of metabolic traits in up to 11,806 individuals by targeted exome sequencing, and follow-up in 39,979 individuals by targeted genotyping, with additional in silico follow up in consortia. We used these data to first compare associations of variants in genes encoding drug targets with the effects of pharmacological manipulation of those targets in clinical trials. We then tested the association those variants with disease outcomes, including coronary heart disease, to predict cardiovascular safety of these agents. A low-frequency missense variant (Ala316Thr;rs10305492) in the gene encoding glucagon-like peptide-1 receptor (GLP1R), the target of GLP1R agonists, was associated with lower fasting glucose and lower T2D risk, consistent with GLP1R agonist therapies. The minor allele was also associated with protection against heart disease, thus providing evidence that GLP1R agonists are not likely to be associated with an unacceptable increase in cardiovascular risk. Our results provide an encouraging signal that these agents may be associated with benefit, a question currently being addressed in randomised controlled trials. Genetic variants associated with metabolic traits and multiple disease outcomes can be used to validate therapeutic targets at an early stage in the drug development process. PMID:27252175

  14. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    NARCIS (Netherlands)

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the

  15. Group-specific 16S rRNA-targeted oligonucleotide probes to identify thermophilic bacteria in marine hydrothermal vents

    NARCIS (Netherlands)

    Harmsen, HJM; Prieur, D; Jeanthon, C

    1997-01-01

    Four 16S rRNA-targeted oligonucleotide probes were designed for the detection of thermophilic members of the domain Bacteria known to thrive in marine hydrothermal systems, We developed and characterized probes encompassing most of the thermophilic members of the genus Bacillus, most species of the

  16. Identifying Candidate Targets of Immune Responses in Zika Virus Based on Homology to Epitopes in Other Flavivirus Species.

    Science.gov (United States)

    Xu, Xiaojun; Vaughan, Kerrie; Weiskopf, Daniela; Grifoni, Alba; Diamond, Michael S; Sette, Alessandro; Peters, Bjoern

    2016-11-15

    The current outbreak of Zika virus has resulted in a massive effort to accelerate the development of ZIKV-specific diagnostics and vaccines. These efforts would benefit greatly from the definition of the specific epitope targets of immune responses in ZIKV, but given the relatively recent emergence of ZIKV as a pandemic threat, few such data are available. We used a large body of epitope data for other Flaviviruses that was available from the IEDB for a comparative analysis against the ZIKV proteome in order to project targets of immune responses in ZIKV. We found a significant level of overlap between known antigenic sites from other Flavivirus proteins with residues on the ZIKV polyprotein. The E and NS1 proteins shared functional antibody epitope sites, whereas regions of T cell reactivity were conserved within NS3 and NS5 for ZIKV.  Discussion: Our epitope based analysis provides guidance for which regions of the ZIKV polyprotein are most likely unique targets of ZIKV-specific antibodies, and which targets in ZIKV are most likely to be cross-reactive with other Flavivirus species. These data may therefore provide insights for the development of antibody- and T cell-based ZIKV-specific diagnostics, therapeutics and prophylaxis.

  17. Emory University: MEDICI (Mining Essentiality Data to Identify Critical Interactions) for Cancer Drug Target Discovery and Development | Office of Cancer Genomics

    Science.gov (United States)

    The CTD2 Center at Emory University has developed a computational methodology to combine high-throughput knockdown data with known protein network topologies to infer the importance of protein-protein interactions (PPIs) for the survival of cancer cells.  Applying these data to the Achilles shRNA results, the CCLE cell line characterizations, and known and newly identified PPIs provides novel insights for potential new drug targets for cancer therapies and identifies important PPI hubs.

  18. A Drosophila model to identify polyamine-drug conjugates that target the polyamine transporter in an intact epithelium.

    Science.gov (United States)

    Tsen, Chung; Iltis, Mark; Kaur, Navneet; Bayer, Cynthia; Delcros, Jean-Guy; von Kalm, Laurence; Phanstiel, Otto

    2008-01-24

    Polyamine transport is elevated in many tumor types, suggesting that toxic polyamine-drug conjugates could be targeted to cancer cells via the polyamine transporter (PAT). We have previously reported the use of Chinese hamster ovary (CHO) cells and its PAT-deficient mutant cell line, CHO-MG, to screen anthracene-polyamine conjugates for their PAT-selective targeting ability. We report here a novel Drosophila-based model for screening anthracene-polyamine conjugates in a developing and intact epithelium ( Drosophila imaginal discs), wherein cell-cell adhesion properties are maintained. Data from the Drosophila assay are consistent with previous results in CHO cells, indicating that the Drosophila epithelium has a PAT with vertebrate-like characteristics. This assay will be of use to medicinal chemists interested in screening drugs that use PAT for cellular entry, and it offers the possibility of genetic dissection of the polyamine transport process, including identification of a Drosophila PAT.

  19. Integration of screening and identifying ligand(s) from medicinal plant extracts based on target recognition by using NMR spectroscopy

    OpenAIRE

    2015-01-01

    Authors: Yalin Tang, Qian Shang, Junfeng Xiang, Qianfan Yang, Qiuju Zhou, Lin Li, Hong Zhang, Qian Li, Hongxia Sun, Aijiao Guan, Wei Jiang & Wei Gai ### Abstract This protocol presents the screening of ligand(s) from medicinal plant extracts based on target recognition by using NMR spectroscopy. A detailed description of sample preparation and analysis process is provided. NMR spectroscopies described here are 1H NMR, diffusion-ordered spectroscopy (DOSY), relaxation-edited NMR, ...

  20. MEDICI: Mining Essentiality Data to Identify Critical Interactions for Cancer Drug Target Discovery and Development | Office of Cancer Genomics

    Science.gov (United States)

    Protein-protein interactions (PPIs) mediate the transmission and regulation of oncogenic signals that are essential to cellular proliferation and survival, and thus represent potential targets for anti-cancer therapeutic discovery. Despite their significance, there is no method to experimentally disrupt and interrogate the essentiality of individual endogenous PPIs. The ability to computationally predict or infer PPI essentiality would help prioritize PPIs for drug discovery and help advance understanding of cancer biology.

  1. Geothermal Energy Potential in Low Enthalpy Areas as a Future Energy Resource: Identifying Feasible Targets, Quebec, Canada, Study Case

    OpenAIRE

    Jacek Majorowicz; Vasile Minea

    2015-01-01

    Heat flow of the sedimentary succession of the Eastern Canada Sedimentary Basins varies from 40 mW/m2 close to the exposed shield in the north to high 60–70 mW/m2 in the southwest–northeast St. Lawrence corridor. As high fluid flow rates are required for a successful geothermal application, the most important targets are deep existing permeable aquifers rather than hard rock, which would need to be fracked. Unfortunately, the ten most populated Québec urban centers are in the areas where the ...

  2. Antibody Responses to NY-ESO-1 in Primary Breast Cancer Identify a Subtype Target for Immunotherapy

    OpenAIRE

    2011-01-01

    The highly immunogenic human tumor antigen NY-ESO-1 (ESO) is a target of choice for anti-cancer immune therapy. In this study, we assessed spontaneous antibody (Ab) responses to ESO in a large cohort of patients with primary breast cancer (BC) and addressed the correlation between the presence of anti-ESO Ab, the expression of ESO in the tumors and their characteristics. We found detectable Ab responses to ESO in 1% of the patients. Tumors from patients with circulating Ab to ESO exhibited co...

  3. Genome-wide Bisulfite Sequencing in Zygotes Identifies Demethylation Targets and Maps the Contribution of TET3 Oxidation

    Directory of Open Access Journals (Sweden)

    Julian R. Peat

    2014-12-01

    Full Text Available Fertilization triggers global erasure of paternal 5-methylcytosine as part of epigenetic reprogramming during the transition from gametic specialization to totipotency. This involves oxidation by TET3, but our understanding of its targets and the wider context of demethylation is limited to a small fraction of the genome. We employed an optimized bisulfite strategy to generate genome-wide methylation profiles of control and TET3-deficient zygotes, using SNPs to access paternal alleles. This revealed that in addition to pervasive removal from intergenic sequences and most retrotransposons, gene bodies constitute a major target of zygotic demethylation. Methylation loss is associated with zygotic genome activation and at gene bodies is also linked to increased transcriptional noise in early development. Our data map the primary contribution of oxidative demethylation to a subset of gene bodies and intergenic sequences and implicate redundant pathways at many loci. Unexpectedly, we demonstrate that TET3 activity also protects certain CpG islands against methylation buildup.

  4. Cell-Internalization SELEX: Method for Identifying Cell-Internalizing RNA Aptamers for Delivering siRNAs to Target Cells

    Science.gov (United States)

    Thiel, William H.; Thiel, Kristina W.; Flenker, Katie S.; Bair, Tom; Dupuy, Adam J.; McNamara, James O.; Miller, Francis J.; Giangrande, Paloma H.

    2015-01-01

    After a decade of work to address cellular uptake, the principal obstacle to RNAi-based therapeutics, there is now well-deserved, renewed optimism about RNAi-based drugs. Phase I and II studies have shown safe, strong, and durable-gene knockdown (80–90 %, lasting for a month after a single injection) and/or clinical benefit in treating several liver pathologies. Although promising, these studies have also highlighted the need for robust delivery techniques to develop RNAi therapeutics for treating other organ systems and diseases. Conjugation of siRNAs to cell-specific, synthetic RNA ligands (aptamers) is being proposed as a viable solution to this problem. While encouraging, the extended use of RNA aptamers as a delivery tool for siRNAs awaits the identification of RNA aptamer sequences capable of targeting and entering the cytoplasm of many different cell types. We describe a cell-based selection process for the rapid identification and characterization of RNA aptamers suited for delivering siRNA drugs into the cytoplasm of target cells. This process, termed “cell-internalization SELEX (Systematic Evolution of Ligands by Exponential Enrichment),” entails the combination of multiple sophisticated technologies, including cell culture-based SELEX procedures, next-generation sequencing (NGS), and novel bioinformatics tools. PMID:25319652

  5. Refining prognosis and identifying targetable pathways for high-risk endometrial cancer; a TransPORTEC initiative

    NARCIS (Netherlands)

    Stelloo, Ellen; Bosse, Tjalling; Nout, Remi A.; MacKay, Helen J.; Church, David N.; Nijman, Hans W.; Leary, Alexandra; Edmondson, Richard J.; Powell, Melanie E.; Crosbie, Emma J.; Kitchener, Henry C.; Mileshkin, Linda; Pollock, Pamela M.; Smit, Vincent T.; Creutzberg, Carien L.

    2015-01-01

    This study aimed to investigate whether molecular analysis can be used to refine risk assessment, direct adjuvant therapy, and identify actionable alterations in high-risk endometrial cancer. TransPORTEC, an international consortium related to the PORTEC3 trial, was established for translational res

  6. Nbs1 ChIP-Seq Identifies Off-Target DNA Double-Strand Breaks Induced by AID in Activated Splenic B Cells.

    Directory of Open Access Journals (Sweden)

    Lyne Khair

    2015-08-01

    Full Text Available Activation-induced cytidine deaminase (AID is required for initiation of Ig class switch recombination (CSR and somatic hypermutation (SHM of antibody genes during immune responses. AID has also been shown to induce chromosomal translocations, mutations, and DNA double-strand breaks (DSBs involving non-Ig genes in activated B cells. To determine what makes a DNA site a target for AID-induced DSBs, we identify off-target DSBs induced by AID by performing chromatin immunoprecipitation (ChIP for Nbs1, a protein that binds DSBs, followed by deep sequencing (ChIP-Seq. We detect and characterize hundreds of off-target AID-dependent DSBs. Two types of tandem repeats are highly enriched within the Nbs1-binding sites: long CA repeats, which can form Z-DNA, and tandem pentamers containing the AID target hotspot WGCW. These tandem repeats are not nearly as enriched at AID-independent DSBs, which we also identified. Msh2, a component of the mismatch repair pathway and important for genome stability, increases off-target DSBs, similar to its effect on Ig switch region DSBs, which are required intermediates during CSR. Most of the off-target DSBs are two-ended, consistent with generation during G1 phase, similar to DSBs in Ig switch regions. However, a minority are one-ended, presumably due to conversion of single-strand breaks to DSBs during replication. One-ended DSBs are repaired by processes involving homologous recombination, including break-induced replication repair, which can lead to genome instability. Off-target DSBs, especially those present during S phase, can lead to chromosomal translocations, deletions and gene amplifications, resulting in the high frequency of B cell lymphomas derived from cells that express or have expressed AID.

  7. Aurora-A identifies early recurrence and poor prognosis and promises a potential therapeutic target in triple negative breast cancer.

    Science.gov (United States)

    Xu, Jie; Wu, Xing; Zhou, Wei-hua; Liu, An-wen; Wu, Jian-bing; Deng, Jin-yun; Yue, Cai-feng; Yang, Shao-bing; Wang, Jing; Yuan, Zhong-yu; Liu, Quentin

    2013-01-01

    Triple negative breast cancer (TNBC) acquires an unfavorable prognosis, emerging as a major challenge for the treatment of breast cancer. In the present study, 122 TNBC patients were subjected to analysis of Aurora-A (Aur-A) expression and survival prognosis. We found that Aur-A high expression was positively associated with initial clinical stage (P = 0.025), the proliferation marker Ki-67 (P = 0.001), and the recurrence rate of TNBC patients (Pprognosis compared with low expression of both Aur-A and Ki-67. Importantly, we further found that Aur-A was overexpressed in TNBC cells, and inhibition of this kinase inhibited cell proliferation and prevented cell migration in TNBC. Our findings demonstrated that Aur-A was a potential therapeutic target for TNBC and inhibition of Aur-A kinase was a promising regimen for TNBC cancer therapy.

  8. Identifying Indices of Happiness and Unhappiness Among Adults With Autism: Potential Targets for Behavioral Assessment and Intervention

    Science.gov (United States)

    Parsons, Marsha B; Bentley, Erik; Inman, Amy; Lattimore, L. Perry

    2012-01-01

    Research is increasingly demonstrating the importance of monitoring indices of happiness as part of behavioral programs for individuals who have severe intellectual disabilities. We evaluated a practitioner-oriented process for identifying and validating individualized indices of this private event among three adults with autism who were nonvocal or minimally vocal. Caregiver surveys were administered to obtain agreement regarding behavior displayed when the individuals were happy and unhappy, as well as situations in which they were likely to experience happiness and unhappiness. Observations corroborated caregiver opinion in that participants displayed more happiness indices in situations reported to be accompanied by happiness, and for the most part, more unhappiness indices in reported unhappiness situations. Subsequent choices by each participant supported the validity of the individualized indices. Results are discussed regarding how identifying happiness indices can be a useful component of behavioral applications, as well as guidelines and cautions regarding use of the indices in routine practice. PMID:23326627

  9. A combined approach to data mining of textual and structured data to identify cancer-related targets

    OpenAIRE

    Adelstein S James; Iyer Lakshmanan K; Pospisil Pavel; Kassis Amin I

    2006-01-01

    Abstract Background We present an effective, rapid, systematic data mining approach for identifying genes or proteins related to a particular interest. A selected combination of programs exploring PubMed abstracts, universal gene/protein databases (UniProt, InterPro, NCBI Entrez), and state-of-the-art pathway knowledge bases (LSGraph and Ingenuity Pathway Analysis) was assembled to distinguish enzymes with hydrolytic activities that are expressed in the extracellular space of cancer cells. Pr...

  10. From C-Reactive Protein to Interleukin-6 to Interleukin-1: Moving Upstream To Identify Novel Targets for Atheroprotection.

    Science.gov (United States)

    Ridker, Paul M

    2016-01-08

    Plasma levels of the inflammatory biomarker high-sensitivity C-reactive protein (hsCRP) predict vascular risk with an effect estimate as large as that of total or high-density lipoprotein cholesterol. Further, randomized trial data addressing hsCRP have been central to understanding the anti-inflammatory effects of statin therapy and have consistently demonstrated on-treatment hsCRP levels to be as powerful a predictor of residual cardiovascular risk as on-treatment levels of low-density lipoprotein cholesterol. Yet, although hsCRP is clinically useful as a biomarker for risk prediction, most mechanistic studies suggest that CRP itself is unlikely to be a target for intervention. Moving upstream in the inflammatory cascade from CRP to interleukin (IL)-6 to IL-1 provides novel therapeutic opportunities for atheroprotection that focus on the central IL-6 signaling system and ultimately on inhibition of the IL-1β-producing NOD-like receptor family pyrin domain containing 3 inflammasome. Cholesterol crystals, neutrophil extracellular traps, atheroprone flow, and local tissue hypoxia activate the NOD-like receptor family pyrin domain containing 3 inflammasome. As such, a unifying concept of hsCRP as a downstream surrogate biomarker for upstream IL-1β activity has emerged. From a therapeutic perspective, small ischemia studies show reductions in acute-phase hsCRP production with the IL-1 receptor antagonist anakinra and the IL-6 receptor blocker tocilizumab. A phase IIb study conducted among diabetic patients at high vascular risk indicates that canakinumab, a human monoclonal antibody that targets IL-1β, markedly reduces plasma levels of IL-6, hsCRP, and fibrinogen with little change in atherogenic lipids. Canakinumab in now being tested as a method to prevent recurrent cardiovascular events in a randomized trial of 10 065 post-myocardial infarction patients with elevated hsCRP that is fully enrolled and due to complete in 2017. Clinical trials using alternative anti

  11. A qualitative study of GP, nurse and practice manager views on using targeted case-finding to identify patients with COPD in primary care.

    Science.gov (United States)

    Summers, Rachael H; Sharmeen, Taniya; Lippiett, Kate; Gillett, Kate; Astles, Carla; Vu, Linh; Stafford-Watson, Mark; Bruton, Anne; Thomas, Mike; Wilkinson, Tom

    2017-08-29

    'Finding the missing millions' with chronic obstructive pulmonary disease became part of the Department of Health strategy for England in 2010. Targeted case-finding within primary care is one potential pro-active strategy, but currently little is known about the views of healthcare professionals on this approach. In this study, 36 healthcare professionals (12 GPs, 14 nurses, and 10 practice managers) from 34 UK practices participated in semi-structured telephone interviews about targeted case-finding. Interviews followed an interview guide, were audio-recorded, transcribed verbatim, coded and analysed using 'Framework Approach'. Most of those interviewed practiced opportunistic case-finding. The main perceived barriers to wider case-finding programmes were the resource implications associated with running such programmes and identifying more chronic obstructive pulmonary disease patients. Financial incentives, support from specialist clinicians, and comprehensive guidance were viewed as facilitators. While targeted case-finding is conceptually accepted by primary care staff, scepticism surrounding (1) the value of identifying those with mild disease and (2) the availability of effective targeted case-finding methods, may lead some to favour an opportunistic approach. Key concerns were a lack of unequivocal evidence for the relative benefits vs. disadvantages of diagnosing patients earlier, and resource constraints in an already over-burdened system. Barriers to practical implementation of case-finding studies may be addressed with financial, human and educational resources, such as additional staff to undertake searches and perform spirometry tests, and practical and educational support from specialist teams. SUPPORT NEEDED TO IDENTIFY THOSE UNDIAGNOSED: Additional staff and resources would facilitate targeted searches for patients showing symptoms of early-stage chronic lung disease. Chronic obstructive pulmonary disease (COPD) costs the UK economy billions of

  12. Aurora-A identifies early recurrence and poor prognosis and promises a potential therapeutic target in triple negative breast cancer.

    Directory of Open Access Journals (Sweden)

    Jie Xu

    Full Text Available Triple negative breast cancer (TNBC acquires an unfavorable prognosis, emerging as a major challenge for the treatment of breast cancer. In the present study, 122 TNBC patients were subjected to analysis of Aurora-A (Aur-A expression and survival prognosis. We found that Aur-A high expression was positively associated with initial clinical stage (P = 0.025, the proliferation marker Ki-67 (P = 0.001, and the recurrence rate of TNBC patients (P<0.001. In TNBC patients with Aur-A high expression, the risk of distant recurrence peaked at the first 3 years and declined rapidly thereafter, whereas patients with Aur-A low expression showed a relatively constant risk of recurrence during the entire follow-up period. Univariate and multivariate analysis showed that overexpression of Aur-A predicted poor overall survival (P = 0.002 and progression-free survival (P = 0.012 in TNBC. Furthermore, overexpression of Aur-A, associated with high Ki-67, predicted an inferior prognosis compared with low expression of both Aur-A and Ki-67. Importantly, we further found that Aur-A was overexpressed in TNBC cells, and inhibition of this kinase inhibited cell proliferation and prevented cell migration in TNBC. Our findings demonstrated that Aur-A was a potential therapeutic target for TNBC and inhibition of Aur-A kinase was a promising regimen for TNBC cancer therapy.

  13. Geothermal Energy Potential in Low Enthalpy Areas as a Future Energy Resource: Identifying Feasible Targets, Quebec, Canada, Study Case

    Directory of Open Access Journals (Sweden)

    Jacek Majorowicz

    2015-07-01

    Full Text Available Heat flow of the sedimentary succession of the Eastern Canada Sedimentary Basins varies from 40 mW/m2 close to the exposed shield in the north to high 60–70 mW/m2 in the southwest–northeast St. Lawrence corridor. As high fluid flow rates are required for a successful geothermal application, the most important targets are deep existing permeable aquifers rather than hard rock, which would need to be fracked. Unfortunately, the ten most populated Québec urban centers are in the areas where the Grenville (Canadian Shield is exposed or at shallow depths with sedimentary cover where temperatures are 30 °C or less. The city of Drummondville will be the exception, as the basement deepens sharply southwest, and higher temperatures reaching >120 °C are expected in the deep Cambrian sedimentary aquifers near a 4–5-km depth. Deep under the area where such sediments could be occurring under Appalachian nappes, temperatures significantly higher than 140 °C are predicted. In parts of the deep basin, temperatures as high as 80 °C–120 °C exist at depths of 3–4 km, mainly southeast of the major geological boundary: the Logan line. There is a large amount of heat resource at such depths to be considered in this area for district heating.

  14. Automated tracking of tumor-stroma morphology in microtissues identifies functional targets within the tumor microenvironment for therapeutic intervention

    Science.gov (United States)

    Åkerfelt, Malin; Bayramoglu, Neslihan; Robinson, Sean; Toriseva, Mervi; Schukov, Hannu-Pekka; Härmä, Ville; Virtanen, Johannes; Sormunen, Raija; Kaakinen, Mika; Kannala, Juho; Eklund, Lauri; Heikkilä, Janne; Nees, Matthias

    2015-01-01

    Cancer-associated fibroblasts (CAFs) constitute an important part of the tumor microenvironment and promote invasion via paracrine functions and physical impact on the tumor. Although the importance of including CAFs into three-dimensional (3D) cell cultures has been acknowledged, computational support for quantitative live-cell measurements of complex cell cultures has been lacking. Here, we have developed a novel automated pipeline to model tumor-stroma interplay, track motility and quantify morphological changes of 3D co-cultures, in real-time live-cell settings. The platform consists of microtissues from prostate cancer cells, combined with CAFs in extracellular matrix that allows biochemical perturbation. Tracking of fibroblast dynamics revealed that CAFs guided the way for tumor cells to invade and increased the growth and invasiveness of tumor organoids. We utilized the platform to determine the efficacy of inhibitors in prostate cancer and the associated tumor microenvironment as a functional unit. Interestingly, certain inhibitors selectively disrupted tumor-CAF interactions, e.g. focal adhesion kinase (FAK) inhibitors specifically blocked tumor growth and invasion concurrently with fibroblast spreading and motility. This complex phenotype was not detected in other standard in vitro models. These results highlight the advantage of our approach, which recapitulates tumor histology and can significantly improve cancer target validation in vitro. PMID:26375443

  15. Microarray-based approach identifies microRNAs and their target functional patterns in polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Boehn Susanne NE

    2008-12-01

    Full Text Available Abstract Background MicroRNAs (miRNAs play key roles in mammalian gene expression and several cellular processes, including differentiation, development, apoptosis and cancer pathomechanisms. Recently the biological importance of primary cilia has been recognized in a number of human genetic diseases. Numerous disorders are related to cilia dysfunction, including polycystic kidney disease (PKD. Although involvement of certain genes and transcriptional networks in PKD development has been shown, not much is known how they are regulated molecularly. Results Given the emerging role of miRNAs in gene expression, we explored the possibilities of miRNA-based regulations in PKD. Here, we analyzed the simultaneous expression changes of miRNAs and mRNAs by microarrays. 935 genes, classified into 24 functional categories, were differentially regulated between PKD and control animals. In parallel, 30 miRNAs were differentially regulated in PKD rats: our results suggest that several miRNAs might be involved in regulating genetic switches in PKD. Furthermore, we describe some newly detected miRNAs, miR-31 and miR-217, in the kidney which have not been reported previously. We determine functionally related gene sets, or pathways to reveal the functional correlation between differentially expressed mRNAs and miRNAs. Conclusion We find that the functional patterns of predicted miRNA targets and differentially expressed mRNAs are similar. Our results suggest an important role of miRNAs in specific pathways underlying PKD.

  16. The Malaria Parasite's Lactate Transporter PfFNT Is the Target of Antiplasmodial Compounds Identified in Whole Cell Phenotypic Screens

    Science.gov (United States)

    Hapuarachchi, Sanduni V.; McConville, Malcolm J.; Martin, Rowena E.; Lehane, Adele M.

    2017-01-01

    In this study the ‘Malaria Box’ chemical library comprising 400 compounds with antiplasmodial activity was screened for compounds that perturb the internal pH of the malaria parasite, Plasmodium falciparum. Fifteen compounds induced an acidification of the parasite cytosol. Two of these did so by inhibiting the parasite’s formate nitrite transporter (PfFNT), which mediates the H+-coupled efflux from the parasite of lactate generated by glycolysis. Both compounds were shown to inhibit lactate transport across the parasite plasma membrane, and the transport of lactate by PfFNT expressed in Xenopus laevis oocytes. PfFNT inhibition caused accumulation of lactate in parasitised erythrocytes, and swelling of both the parasite and parasitised erythrocyte. Long-term exposure of parasites to one of the inhibitors gave rise to resistant parasites with a mutant form of PfFNT that showed reduced inhibitor sensitivity. This study provides the first evidence that PfFNT is a druggable antimalarial target. PMID:28178359

  17. Development and exploitation of a novel mutant androgen receptor modelling strategy to identify new targets for advanced prostate cancer therapy.

    Science.gov (United States)

    O'Neill, Daniel; Jones, Dominic; Wade, Mark; Grey, James; Nakjang, Sirintra; Guo, Wenrui; Cork, David; Davies, Barry R; Wedge, Steve R; Robson, Craig N; Gaughan, Luke

    2015-09-22

    The persistence of androgen receptor (AR) signalling in castrate-resistant prostate cancer (CRPC) highlights the unmet clinical need for the development of more effective AR targeting therapies. A key mechanism of therapy-resistance is by selection of AR mutations that convert anti-androgens to agonists enabling the retention of androgenic signalling in CRPC. To improve our understanding of these receptors in advanced disease we developed a physiologically-relevant model to analyse the global functionality of AR mutants in CRPC. Using the bicalutamide-activated AR(W741L/C) mutation as proof of concept, we demonstrate that this mutant confers an androgenic-like signalling programme and growth promoting phenotype in the presence of bicalutamide. Transcriptomic profiling of AR(W741L) highlighted key genes markedly up-regulated by the mutant receptor, including TIPARP, RASD1 and SGK1. Importantly, SGK1 expression was found to be highly expressed in the KUCaP xenograft model and a CRPC patient biopsy sample both of which express the bicalutamide-activated receptor mutant. Using an SGK1 inhibitor, AR(W741L) transcriptional and growth promoting activity was reduced indicating that exploiting functional distinctions between receptor isoforms in our model may provide new and effective therapies for CRPC patients.

  18. Immunoproteomically identified GBAA_0345, alkyl hydroperoxide reductase subunit C is a potential target for multivalent anthrax vaccine.

    Science.gov (United States)

    Kim, Yeon Hee; Kim, Kyung Ae; Kim, Yu-Ri; Choi, Min Kyung; Kim, Hye Kyeong; Choi, Ki Ju; Chun, Jeong-Hoon; Cha, Kiweon; Hong, Kee-Jong; Lee, Na Gyong; Yoo, Cheon-Kwon; Oh, Hee-Bok; Kim, Tae Sung; Rhie, Gi-eun

    2014-01-01

    Anthrax is caused by the spore-forming bacterium Bacillus anthracis, which has been used as a weapon for bioterrorism. Although current vaccines are effective, they involve prolonged dose regimens and often cause adverse reactions. High rates of mortality associated with anthrax have made the development of an improved vaccine a top priority. To identify novel vaccine candidates, we applied an immunoproteomics approach. Using sera from convalescent guinea pigs or from human patients with anthrax, we identified 34 immunogenic proteins from the virulent B. anthracis H9401. To evaluate vaccine candidates, six were expressed as recombinant proteins and tested in vivo. Two proteins, rGBAA_0345 (alkyl hydroperoxide reductase subunit C) and rGBAA_3990 (malonyl CoA-acyl carrier protein transacylase), have afforded guinea pigs partial protection from a subsequent virulent-spore challenge. Moreover, combined vaccination with rGBAA_0345 and rPA (protective antigen) exhibited an enhanced ability to protect against anthrax mortality. Finally, we demonstrated that GBAA_0345 localizes to anthrax spores and bacilli. Our results indicate that rGBAA_0345 may be a potential component of a multivalent anthrax vaccine, as it enhances the efficacy of rPA vaccination. This is the first time that sera from patients with anthrax have been used to interrogate the proteome of virulent B. anthracis vegetative cells.

  19. Giardia duodenalis genotypes in domestic and wild animals from Romania identified by PCR-RFLP targeting the gdh gene.

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    Adriana, Gyӧrke; Zsuzsa, Kalmár; Mirabela Oana, Dumitrache; Mircea, Gherman Călin; Viorica, Mircean

    2016-02-15

    Sixty Giardia duodenalis isolates from domestic (n=49) and wild (n=11) animals (dogs, cats, deers, wolves, raccoon dog and muskrat) were analysed by PCR-RFLP at glutamate dehydrogenase locus (gdh). The isolates were obtained from positive feces samples for Giardia cysts analysed by flotation technique with saturated sodium chloride solution (specific gravity 1.28). Three G. duodenalis genotypes were identified: C (10/60; 16.7%); D (42/60; 70.0%); and E (7/60; 11.7%). In dogs all three genotypes were found, with the following prevalences: 76.9% genotype D (30/39); 23.1% C (9/39); 2.6% genotype E (1/39). One dog was co-infected with C and D genotypes. In cats we identified only G. duodenalis genotype D. Wolves and raccoon dog harbored infection with G. duodenalis genotype D, deers with E type and muskrat C type. This is the first study regarding genotyping of G. duodenalis in cats and wild animals from Romania. To the best of our knowledge, this is the first report of assemblages E in roe deers; assemblage C in wolves and muskrat; and assemblage D in raccoon dog.

  20. Targeted next-generation sequencing of cancer genes identified frequent TP53 and ATRX mutations in leiomyosarcoma.

    Science.gov (United States)

    Yang, Ching-Yao; Liau, Jau-Yu; Huang, Wei-Ju; Chang, Yu-Ting; Chang, Ming-Chu; Lee, Jen-Chieh; Tsai, Jia-Huei; Su, Yi-Ning; Hung, Chia-Cheng; Jeng, Yung-Ming

    2015-01-01

    Leiomyosarcoma is an aggressive soft tissue sarcoma with poor patient survival. The genetic changes of leiomyosarcoma remain to be discovered. In this study, we analyzed the genetic changes of 44 cancer-related genes by using next-generation sequencing in 54 leiomyosarcomas. We identified TP53 mutations in 19 of the 54 tumors (35%) and ATRX mutations in 9 of the 54 tumors (17%). The TP53-mutated leiomyosarcomas were limited to female patients (P = 0.006). All but 2 of the TP53-mutated leiomyosarcomas were located in the uterus (n = 11) or retroperitoneum (n = 6). The ATRX mutations were associated with poorly differentiated leiomyosarcomas (P = 0.028) and the presence of tumor necrosis (P = 0.015). Kaplan-Meier survival analysis showed that patients with ATRX-mutated leiomyosarcomas had worse overall survival than did patients with ATRX-wild-type leiomyosarcomas. All of the ATRX-mutated leiomyosarcomas showed the alternative lengthening of telomere phenotype. The ATRX mutations did not correlate with ATRX protein expression, as detected using immunohistochemistry. In conclusion, we identified loss of function of the p53 and ATRX pathways being the main mechanisms for leiomyosarcomas. The molecular mechanisms may provide new opportunities to treat these aggressive neoplasms.

  1. An in vivo reporter assay for sRNA-directed gene control in Gram-positive bacteria: identifying a novel sRNA target in Staphylococcus aureus

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    Ivain, Lorraine; Bordeau, Valérie; Eyraud, Alex; Hallier, Marc; Dreano, Stéphane; Tattevin, Pierre

    2017-01-01

    Abstract Bacterial small regulatory RNAs (sRNAs) play a major role in the regulation of various cellular functions. Most sRNAs interact with mRNA targets via an antisense mechanism, modifying their translation and/or degradation. Despite considerable progresses in discovering sRNAs in Gram-positive bacteria, their functions, for the most part, are unknown. This is mainly due to difficulties in identifying their targets. To aid in the identification of sRNA targets in Gram-positive bacteria, we set up an in vivo method for fast analysis of sRNA-mediated post-transcriptional control at the 5΄ regions of target mRNAs. The technology is based on the co-expression of an sRNA and a 5΄ sequence of an mRNA target fused to a green fluorescent protein (GFP) reporter. The system was challenged on Staphylococcus aureus, an opportunistic Gram-positive pathogen. We analyzed several established sRNA–mRNA interactions, and in addition, we identified the ecb mRNA as a novel target for SprX2 sRNA. Using our in vivo system in combination with in vitro experiments, we demonstrated that SprX2 uses an antisense mechanism to prevent ecb mRNA translation initiation. Furthermore, we used our reporter assay to validate sRNA regulations in other Gram-positive organisms, Bacillus subtilis and Listeria monocytogenes. Overall, our method is broadly applicable to challenge the predicted sRNA–mRNA interactions in Gram-positive bacteria. PMID:28369640

  2. A Sensitive in Vitro High-Throughput Screen To Identify Pan-filoviral Replication Inhibitors Targeting the VP35–NP Interface

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    Liu, Gai; Nash, Peter J.; Johnson, Britney; Pietzsch, Colette; Ilagan, Ma. Xenia G.; Bukreyev, Alexander; Basler, Christopher F.; Bowlin, Terry L.; Moir, Donald T.; Leung, Daisy W.; Amarasinghe, Gaya K.

    2017-01-24

    The 2014 Ebola outbreak in West Africa, the largest outbreak on record, highlighted the need for novel approaches to therapeutics targeting Ebola virus (EBOV). Within the EBOV replication complex, the interaction between polymerase cofactor, viral protein 35 (VP35), and nucleoprotein (NP) is critical for viral RNA synthesis. We recently identified a peptide at the N-terminus of VP35 (termed NPBP) that is sufficient for interaction with NP and suppresses EBOV replication, suggesting that the NPBP binding pocket can serve as a potential drug target. Here we describe the development and validation of a sensitive high-throughput screen (HTS) using a fluorescence polarization assay. Initial hits from this HTS include the FDA-approved compound tolcapone, whose potency against EBOV infection was validated in a nonfluorescent secondary assay. High conservation of the NP–VP35 interface among filoviruses suggests that this assay has the capacity to identify pan-filoviral inhibitors for development as antivirals.

  3. Screening of Conditionally Reprogrammed Patient-Derived Carcinoma Cells Identifies ERCC3-MYC Interactions as a Target in Pancreatic Cancer.

    Science.gov (United States)

    Beglyarova, Natalya; Banina, Eugenia; Zhou, Yan; Mukhamadeeva, Ramilia; Andrianov, Grigorii; Bobrov, Egor; Lysenko, Elena; Skobeleva, Natalya; Gabitova, Linara; Restifo, Diana; Pressman, Max; Serebriiskii, Ilya G; Hoffman, John P; Paz, Keren; Behrens, Diana; Khazak, Vladimir; Jablonski, Sandra A; Golemis, Erica A; Weiner, Louis M; Astsaturov, Igor

    2016-12-15

    Even when diagnosed prior to metastasis, pancreatic ductal adenocarcinoma (PDAC) is a devastating malignancy with almost 90% lethality, emphasizing the need for new therapies optimally targeting the tumors of individual patients. We first developed a panel of new physiologic models for study of PDAC, expanding surgical PDAC tumor samples in culture using short-term culture and conditional reprogramming with the Rho kinase inhibitor Y-27632, and creating matched patient-derived xenografts (PDX). These were evaluated for sensitivity to a large panel of clinical agents, and promising leads further evaluated mechanistically. Only a small minority of tested agents was cytotoxic in minimally passaged PDAC cultures in vitro Drugs interfering with protein turnover and transcription were among most cytotoxic. Among transcriptional repressors, triptolide, a covalent inhibitor of ERCC3, was most consistently effective in vitro and in vivo causing prolonged complete regression in multiple PDX models resistant to standard PDAC therapies. Importantly, triptolide showed superior activity in MYC-amplified PDX models and elicited rapid and profound depletion of the oncoprotein MYC, a transcriptional regulator. Expression of ERCC3 and MYC was interdependent in PDACs, and acquired resistance to triptolide depended on elevated ERCC3 and MYC expression. The Cancer Genome Atlas analysis indicates ERCC3 expression predicts poor prognosis, particularly in CDKN2A-null, highly proliferative tumors. This provides initial preclinical evidence for an essential role of MYC-ERCC3 interactions in PDAC, and suggests a new mechanistic approach for disruption of critical survival signaling in MYC-dependent cancers. Clin Cancer Res; 22(24); 6153-63. ©2016 AACR. ©2016 American Association for Cancer Research.

  4. Transcriptomic and behavioural characterisation of a mouse model of burn pain identify the cholecystokinin 2 receptor as an analgesic target.

    Science.gov (United States)

    Yin, Kathleen; Deuis, Jennifer R; Lewis, Richard J; Vetter, Irina

    2016-01-01

    Burn injury is a cause of significant mortality and morbidity worldwide and is frequently associated with severe and long-lasting pain that remains difficult to manage throughout recovery. We characterised a mouse model of burn-induced pain using pharmacological and transcriptomic approaches. Mechanical allodynia elicited by burn injury was partially reversed by meloxicam (5 mg/kg), gabapentin (100 mg/kg) and oxycodone (3 and 10 mg/kg), while thermal allodynia and gait abnormalities were only significantly improved by amitriptyline (3 mg/kg) and oxycodone (10 mg/kg). The need for relatively high opioid doses to elicit analgesia suggested a degree of opioid resistance, similar to that shown clinically in burn patients. We thus assessed the gene expression changes in dorsal root ganglion neurons and pathophysiological mechanisms underpinning burn injury-induced pain using a transcriptomic approach. Burn injury was associated with significantly increased expression of genes associated with axon guidance, neuropeptide signalling, behavioural defence response and extracellular signalling, confirming a mixed neuropathic and inflammatory aetiology. Notably, among the pain-related genes that were upregulated post-injury was the cholecystokinin 2 receptor (Cckbr), a G protein-coupled receptor known as a pain target involved in reducing opioid effectiveness. Indeed, the clinically used cholecystokinin receptor antagonist proglumide (30 mg/kg) was effective at reversing mechanical allodynia, with additional analgesia evident in combination with low-dose oxycodone (1 mg/kg), including significant reversal of thermal allodynia. These findings highlight the complex pathophysiological mechanisms underpinning burn injury-induced pain and suggest that cholecystokinin-2 receptor antagonists may be useful clinically as adjuvants to decrease opioid requirements and improve analgesic management.

  5. Runx1 regulation of Pu.1 corepressor/coactivator exchange identifies specific molecular targets for leukemia differentiation therapy.

    Science.gov (United States)

    Gu, Xiaorong; Hu, Zhenbo; Ebrahem, Quteba; Crabb, John S; Mahfouz, Reda Z; Radivoyevitch, Tomas; Crabb, John W; Saunthararajah, Yogen

    2014-05-23

    Gene activation requires cooperative assembly of multiprotein transcription factor-coregulator complexes. Disruption to cooperative assemblage could underlie repression of tumor suppressor genes in leukemia cells. Mechanisms of cooperation and its disruption were therefore examined for PU.1 and RUNX1, transcription factors that cooperate to activate hematopoietic differentiation genes. PU.1 is highly expressed in leukemia cells, whereas RUNX1 is frequently inactivated by mutation or translocation. Thus, coregulator interactions of Pu.1 were examined by immunoprecipitation coupled with tandem mass spectrometry/Western blot in wild-type and Runx1-deficient hematopoietic cells. In wild-type cells, the NuAT and Baf families of coactivators coimmunoprecipitated with Pu.1. Runx1 deficiency produced a striking switch to Pu.1 interaction with the Dnmt1, Sin3A, Nurd, CoRest, and B-Wich corepressor families. Corepressors of the Polycomb family, which are frequently inactivated by mutation or deletion in myeloid leukemia, did not interact with Pu.1. The most significant gene ontology association of Runx1-Pu.1 co-bound genes was with macrophages, therefore, functional consequences of altered corepressor/coactivator exchange were examined at Mcsfr, a key macrophage differentiation gene. In chromatin immunoprecipitation analyses, high level Pu.1 binding to the Mcsfr promoter was not decreased by Runx1 deficiency. However, the Pu.1-driven shift from histone repression to activation marks at this locus, and terminal macrophage differentiation, were substantially diminished. DNMT1 inhibition, but not Polycomb inhibition, in RUNX1-translocated leukemia cells induced terminal differentiation. Thus, RUNX1 and PU.1 cooperate to exchange corepressors for coactivators, and the specific corepressors recruited to PU.1 as a consequence of RUNX1 deficiency could be rational targets for leukemia differentiation therapy.

  6. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

    Science.gov (United States)

    Yu, Chunxiao; Lopez, Carlos A; Hu, Han; Xia, Yu; Freedman, David S; Reddington, Alexander P; Daaboul, George G; Unlü, M Selim; Genco, Caroline Attardo

    2014-01-01

    The Ferric uptake regulatory protein (Fur) is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS), to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  7. Health-related quality of life among veterans in addictions treatment: identifying behavioral targets for future intervention

    Science.gov (United States)

    Oppezzo, Marily A.; Michalek, Anne K.; Delucchi, Kevin; Baiocchi, Michael T. M.; Barnett, Paul G.

    2016-01-01

    Background US veterans report lower health-related quality of life (HRQoL) relative to the general population. Identifying behavioral factors related to HRQoL that are malleable to change may inform interventions to improve well-being in this vulnerable group. Purpose The current study sought to characterize HRQoL in a largely male sample of veterans in addictions treatment, both in relation to US norms and in association with five recommended health behavior practices: regularly exercising, managing stress, having good sleep hygiene, consuming fruits and vegetables, and being tobacco free. Methods We assessed HRQoL with 250 veterans in addictions treatment (96 % male, mean age 53, range 24–77) using scales from four validated measures. Data reduction methods identified two principal components reflecting physical and mental HRQoL. Model testing of HRQoL associations with health behaviors adjusted for relevant demographic and treatment-related covariates. Results Compared to US norms, the sample had lower HRQoL scores. Better psychological HRQoL was associated with higher subjective social standing, absence of pain or trauma, lower alcohol severity, and monotonically with the sum of health behaviors (all p management, and sleep hygiene. Regular exercise also related to better physical HRQoL. The models explained >40 % of the variance in HRQoL. Conclusions Exercise, sleep hygiene, and stress management are strongly associated with HRQoL among veterans in addictions treatment. Future research is needed to test the effect of interventions for improving well-being in this high-risk group. PMID:26886926

  8. Can the microRNA expression profile help to identify novel targets for zoledronic acid in breast cancer?

    Science.gov (United States)

    Insalaco, Lavinia; Incorvaia, Lorena; Barraco, Nadia; Castiglia, Marta; Rizzo, Sergio; Santini, Daniele; Giordano, Antonio; Castorina, Sergio; Russo, Antonio

    2016-01-01

    Zoledronic acid (ZOL), belonging to third generation bisphosphonate family, is a potent inhibitor of osteoclast-mediated bone resorption, widely used to effectively prevent osteolysis in breast cancer patients who develop bone metastases. Low doses of ZOL have been shown to exhibit a direct anticancer role, by inhibiting cell adhesion, invasion, cytoskeleton remodelling and proliferation in MCF-7 breast cancer cells. In order to identify the molecular mechanisms and signaling pathways underlying the anticancer activity exerted by ZOL, we analyzed for the first time the microRNA expression profile in breast cancer cells. A large-scale microarray analysis of 377 miRNAs was performed on MCF7 cells treated with 10 μM ZOL for 24 h compared to untreated cells. Furthermore, the expression of specific ZOL-induced miRNAs was analyzed in MCF-7 and SkBr3 cells through Real-time PCR. Low-dose treatment with ZOL significantly altered expression of 54 miRNAs. Nine upregulated and twelve downregulated miRNAs have been identified after 24 h of treatment. Also, ZOL induced expression of 11 specific miRNAs and silenced expression of 22 miRNAs. MiRNA data analysis revealed the involvement of differentially expressed miRNAs in PI3K/Akt, MAPK, Wnt, TGF-β, Jak-STAT and mTOR signaling pathways, and regulation of actin cytoskeleton. Our results have been shown to be perfectly coherent with the recent findings reported in literature concerning changes in expression of some miRNAs involved in bone metastasis formation, progression, therapy resistance in breast cancer. In conclusion, this data supports the hypothesis that ZOL-induced modification of the miRNA expression profile contributes to the anticancer efficacy of this agent. PMID:27081088

  9. Validation of a Non-Targeted LC-MS Approach for Identifying Ancient Proteins: Method Development on Bone to Improve Artifact Residue Analysis

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    Andrew Barker

    2015-09-01

    Full Text Available Identification of protein residues from prehistoric cooking pottery using mass spectrometry is challenging because proteins are removed from original tissues, are degraded from cooking, may be poorly preserved due to diagenesis, and occur in a palimpsest of exogenous soil proteins. In contrast, bone proteins are abundant and well preserved. This research is part of a larger method-development project for innovation and improvement of liquid chromatography – mass spectrometry analysis of protein residues from cooking pottery; here we validate the potential of our extraction and characterization approach via application to ancient bone proteins. Because of its preservation potential for proteins and given that our approach is destructive, ancient bone identified via skeletal morphology represents an appropriate verification target. Proteins were identified from zooarchaeological turkey (Meleagris gallopavo Linnaeus Phasianidae, rabbit (Lagomorpha, and squirrel (Sciuridae remains excavated from ancient pueblo archaeological sites in southwestern Colorado using a non-targeted LC-MS/MS approach. The data have been deposited to the ProteomeXchange Consortium with the dataset identifier PXD002440. Improvement of highly sensitive targeted LC-MS/MS approaches is an avenue for future method development related to the study of protein residues from artifacts such as stone tools and pottery.

  10. Integrated compound profiling screens identify the mitochondrial electron transport chain as the molecular target of the natural products manassantin, sesquicillin, and arctigenin.

    Science.gov (United States)

    Lai, Kevin; Selinger, Douglas W; Solomon, Jonathan M; Wu, Hua; Schmitt, Esther; Serluca, Fabrizio C; Curtis, Daniel; Benson, John D

    2013-01-18

    Phenotypic compound screens can be used to identify novel targets in signaling pathways and disease processes, but the usefulness of these screens depends on the ability to quickly determine the target and mechanism of action of the molecules identified as hits. One fast route to discovering the mechanism of action of a compound is to profile its properties and to match this profile with those of compounds of known mechanism of action. In this work, the Novartis collection of over 12,000 pure natural products was screened for effects on early zebrafish development. The largest phenotypic class of hits, which caused developmental arrest without necrosis, contained known electron transport chain inhibitors and many compounds of unknown mechanism of action. High-throughput transcriptional profiling revealed that these compounds are mechanistically related to one another. Metabolic and biochemical assays confirmed that all of the molecules that induced developmental arrest without necrosis inhibited the electron transport chain. These experiments demonstrate that the electron transport chain is the target of the natural products manassantin, sesquicillin, and arctigenin. The overlap between the zebrafish and transcriptional profiling screens was not perfect, indicating that multiple profiling screens are necessary to fully characterize molecules of unknown function. Together, zebrafish screening and transcriptional profiling represent sensitive and scalable approaches for identifying bioactive compounds and elucidating their mechanism of action.

  11. Synthetic lethal genetic interactions that decrease somatic cell proliferation in Caenorhabditis elegans identify the alternative RFC CTF18 as a candidate cancer drug target.

    Science.gov (United States)

    McLellan, Jessica; O'Neil, Nigel; Tarailo, Sanja; Stoepel, Jan; Bryan, Jennifer; Rose, Ann; Hieter, Philip

    2009-12-01

    Somatic mutations causing chromosome instability (CIN) in tumors can be exploited for selective killing of cancer cells by knockdown of second-site genes causing synthetic lethality. We tested and statistically validated synthetic lethal (SL) interactions between mutations in six Saccharomyces cerevisiae CIN genes orthologous to genes mutated in colon tumors and five additional CIN genes. To identify which SL interactions are conserved in higher organisms and represent potential chemotherapeutic targets, we developed an assay system in Caenorhabditis elegans to test genetic interactions causing synthetic proliferation defects in somatic cells. We made use of postembryonic RNA interference and the vulval cell lineage of C. elegans as a readout for somatic cell proliferation defects. We identified SL interactions between members of the cohesin complex and CTF4, RAD27, and components of the alternative RFC(CTF18) complex. The genetic interactions tested are highly conserved between S. cerevisiae and C. elegans and suggest that the alternative RFC components DCC1, CTF8, and CTF18 are ideal therapeutic targets because of their mild phenotype when knocked down singly in C. elegans. Furthermore, the C. elegans assay system will contribute to our knowledge of genetic interactions in a multicellular animal and is a powerful approach to identify new cancer therapeutic targets.

  12. Native human autoantibodies targeting GIPC1 identify differential expression in malignant tumors of the breast and ovary

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    Yavelsky Victoria

    2008-08-01

    Full Text Available Abstract Background We have been studying the native humoral immune response to cancer and have isolated a library of fully human autoantibodies to a variety of malignancies. We previously described the isolation and characterization of two fully human monoclonal antibodies, 27.F7 and 27.B1, from breast cancer patients that target the protein known as GIPC1, an accessory PDZ-domain binding protein involved in regulation of G-protein signaling. Human monoclonal antibodies, 27.F7 and 27.B1, to GIPC1 demonstrate specific binding to malignant breast cancer tissue with no reactivity with normal breast tissue. Methods The current study employs cELISA, flow cytometry, Western blot analysis as well as immunocytochemistry, and immunohistochemistry. Data is analyzed statistically with the Fisher one-tail and two-tail tests for two independent samples. Results By screening several other cancer cell lines with 27.F7 and 27.B1 we found consistently strong staining of other human cancer cell lines including SKOV-3 (an ovarian cancer cell line. To further clarify the association of GIPC1 with breast and ovarian cancer we carefully studied 27.F7 and 27.B1 using immunocytochemical and immunohistochemical techniques. An immunohistochemical study of normal ovarian tissue, benign, borderline and malignant ovarian serous tumors, and different types of breast cancer revealed high expression of GIPC1 protein in neoplastic cells. Interestingly, antibodies 27.F7 and 27.B1 demonstrate differential staining of borderline ovarian tumors. Examination of different types of breast cancer demonstrates that the level of GIPC1 expression depends on tumor invasiveness and displays a higher expression than in benign tumors. Conclusion The present pilot study demonstrates that the GIPC1 protein is overexpressed in ovarian and breast cancer, which may provide an important diagnostic and prognostic marker and will constitute the basis for further study of the role that this protein

  13. Recurrent targeted genes of hepatitis B virus in the liver cancer genomes identified by a next-generation sequencing-based approach.

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    Dong Ding

    Full Text Available Integration of the viral DNA into host chromosomes was found in most of the hepatitis B virus (HBV-related hepatocellular carcinomas (HCCs. Here we devised a massive anchored parallel sequencing (MAPS method using next-generation sequencing to isolate and sequence HBV integrants. Applying MAPS to 40 pairs of HBV-related HCC tissues (cancer and adjacent tissues, we identified 296 HBV integration events corresponding to 286 unique integration sites (UISs with precise HBV-Human DNA junctions. HBV integration favored chromosome 17 and preferentially integrated into human transcript units. HBV targeted genes were enriched in GO terms: cAMP metabolic processes, T cell differentiation and activation, TGF beta receptor pathway, ncRNA catabolic process, and dsRNA fragmentation and cellular response to dsRNA. The HBV targeted genes include 7 genes (PTPRJ, CNTN6, IL12B, MYOM1, FNDC3B, LRFN2, FN1 containing IPR003961 (Fibronectin, type III domain, 7 genes (NRG3, MASP2, NELL1, LRP1B, ADAM21, NRXN1, FN1 containing IPR013032 (EGF-like region, conserved site, and three genes (PDE7A, PDE4B, PDE11A containing IPR002073 (3', 5'-cyclic-nucleotide phosphodiesterase. Enriched pathways include hsa04512 (ECM-receptor interaction, hsa04510 (Focal adhesion, and hsa04012 (ErbB signaling pathway. Fewer integration events were found in cancers compared to cancer-adjacent tissues, suggesting a clonal expansion model in HCC development. Finally, we identified 8 genes that were recurrent target genes by HBV integration including fibronectin 1 (FN1 and telomerase reverse transcriptase (TERT1, two known recurrent target genes, and additional novel target genes such as SMAD family member 5 (SMAD5, phosphatase and actin regulator 4 (PHACTR4, and RNA binding protein fox-1 homolog (C. elegans 1 (RBFOX1. Integrating analysis with recently published whole-genome sequencing analysis, we identified 14 additional recurrent HBV target genes, greatly expanding the HBV recurrent target list

  14. Applying the Theoretical Domains Framework to identify barriers and targeted interventions to enhance nurses' use of electronic medication management systems in two Australian hospitals.

    Science.gov (United States)

    Debono, Deborah; Taylor, Natalie; Lipworth, Wendy; Greenfield, David; Travaglia, Joanne; Black, Deborah; Braithwaite, Jeffrey

    2017-03-27

    Medication errors harm hospitalised patients and increase health care costs. Electronic Medication Management Systems (EMMS) have been shown to reduce medication errors. However, nurses do not always use EMMS as intended, largely because implementation of such patient safety strategies requires clinicians to change their existing practices, routines and behaviour. This study uses the Theoretical Domains Framework (TDF) to identify barriers and targeted interventions to enhance nurses' appropriate use of EMMS in two Australian hospitals. This qualitative study draws on in-depth interviews with 19 acute care nurses who used EMMS. A convenience sampling approach was used. Nurses working on the study units (N = 6) in two hospitals were invited to participate if available during the data collection period. Interviews inductively explored nurses' experiences of using EMMS (step 1). Data were analysed using the TDF to identify theory-derived barriers to nurses' appropriate use of EMMS (step 2). Relevant behaviour change techniques (BCTs) were identified to overcome key barriers to using EMMS (step 3) followed by the identification of potential literature-informed targeted intervention strategies to operationalise the identified BCTs (step 4). Barriers to nurses' use of EMMS in acute care were represented by nine domains of the TDF. Two closely linked domains emerged as major barriers to EMMS use: Environmental Context and Resources (availability and properties of computers on wheels (COWs); technology characteristics; specific contexts; competing demands and time pressure) and Social/Professional Role and Identity (conflict between using EMMS appropriately and executing behaviours critical to nurses' professional role and identity). The study identified three potential BCTs to address the Environmental Context and Resources domain barrier: adding objects to the environment; restructuring the physical environment; and prompts and cues. Seven BCTs to address Social

  15. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

    Science.gov (United States)

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

  16. In vivo conditions to identify Prkci phosphorylation targets using the analog-sensitive kinase method in zebrafish.

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    Elena Cibrián Uhalte

    Full Text Available Protein kinase C iota is required for various cell biological processes including epithelial tissue polarity and organ morphogenesis. To gain mechanistic insight into different roles of this kinase, it is essential to identify specific substrate proteins in their cellular context. The analog-sensitive kinase method provides a powerful tool for the identification of kinase substrates under in vivo conditions. However, it has remained a major challenge to establish screens based on this method in multicellular model organisms. Here, we report the methodology for in vivo conditions using the analog-sensitive kinase method in a genetically-tractable vertebrate model organism, the zebrafish. With this approach, kinase substrates can uniquely be labeled in the developing zebrafish embryo using bulky ATPγS analogs which results in the thiophosphorylation of substrates. The labeling of kinase substrates with a thiophosphoester epitope differs from phosphoesters that are generated by all other kinases and allows for an enrichment of thiophosphopeptides by immunoaffinity purification. This study provides the foundation for using the analog-sensitive kinase method in the context of complex vertebrate development, physiology, or disease.

  17. Chemical screening identifies ROCK as a target for recovering mitochondrial function in Hutchinson-Gilford progeria syndrome.

    Science.gov (United States)

    Kang, Hyun Tae; Park, Joon Tae; Choi, Kobong; Choi, Hyo Jei Claudia; Jung, Chul Won; Kim, Gyu Ree; Lee, Young-Sam; Park, Sang Chul

    2017-03-19

    Hutchinson-Gilford progeria syndrome (HGPS) constitutes a genetic disease wherein an aging phenotype manifests in childhood. Recent studies indicate that reactive oxygen species (ROS) play important roles in HGPS phenotype progression. Thus, pharmacological reduction in ROS levels has been proposed as a potentially effective treatment for patient with this disorder. In this study, we performed high-throughput screening to find compounds that could reduce ROS levels in HGPS fibroblasts and identified rho-associated protein kinase (ROCK) inhibitor (Y-27632) as an effective agent. To elucidate the underlying mechanism of ROCK in regulating ROS levels, we performed a yeast two-hybrid screen and discovered that ROCK1 interacts with Rac1b. ROCK activation phosphorylated Rac1b at Ser71 and increased ROS levels by facilitating the interaction between Rac1b and cytochrome c. Conversely, ROCK inactivation with Y-27632 abolished their interaction, concomitant with ROS reduction. Additionally, ROCK activation resulted in mitochondrial dysfunction, whereas ROCK inactivation with Y-27632 induced the recovery of mitochondrial function. Furthermore, a reduction in the frequency of abnormal nuclear morphology and DNA double-strand breaks was observed along with decreased ROS levels. Thus, our study reveals a novel mechanism through which alleviation of the HGPS phenotype is mediated by the recovery of mitochondrial function upon ROCK inactivation.

  18. Novel mitochondria-targeted heat-soluble proteins identified in the anhydrobiotic Tardigrade improve osmotic tolerance of human cells.

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    Sae Tanaka

    Full Text Available Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called "anhydrobiosis". Late Embryogenesis Abundant (LEA proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble and SAHS (Secretory Abundant Heat Soluble proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble, as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments.

  19. Novel Mitochondria-Targeted Heat-Soluble Proteins Identified in the Anhydrobiotic Tardigrade Improve Osmotic Tolerance of Human Cells

    Science.gov (United States)

    Tanaka, Sae; Tanaka, Junko; Miwa, Yoshihiro; Horikawa, Daiki D.; Katayama, Toshiaki; Arakawa, Kazuharu; Toyoda, Atsushi; Kubo, Takeo; Kunieda, Takekazu

    2015-01-01

    Tardigrades are able to tolerate almost complete dehydration through transition to a metabolically inactive state, called “anhydrobiosis”. Late Embryogenesis Abundant (LEA) proteins are heat-soluble proteins involved in the desiccation tolerance of many anhydrobiotic organisms. Tardigrades, Ramazzottius varieornatus, however, express predominantly tardigrade-unique heat-soluble proteins: CAHS (Cytoplasmic Abundant Heat Soluble) and SAHS (Secretory Abundant Heat Soluble) proteins, which are secreted or localized in most intracellular compartments, except the mitochondria. Although mitochondrial integrity is crucial to ensure cellular survival, protective molecules for mitochondria have remained elusive. Here, we identified two novel mitochondrial heat-soluble proteins, RvLEAM and MAHS (Mitochondrial Abundant Heat Soluble), as potent mitochondrial protectants from Ramazzottius varieornatus. RvLEAM is a group3 LEA protein and immunohistochemistry confirmed its mitochondrial localization in tardigrade cells. MAHS-green fluorescent protein fusion protein localized in human mitochondria and was heat-soluble in vitro, though no sequence similarity with other known proteins was found, and one region was conserved among tardigrades. Furthermore, we demonstrated that RvLEAM protein as well as MAHS protein improved the hyperosmotic tolerance of human cells. The findings of the present study revealed that tardigrade mitochondria contain at least two types of heat-soluble proteins that might have protective roles in water-deficient environments. PMID:25675104

  20. Systematic screening of isogenic cancer cells identifies DUSP6 as context-specific synthetic lethal target in melanoma

    DEFF Research Database (Denmark)

    Wittig-Blaich, Stephanie; Wittig, Rainer; Schmidt, Steffen

    2017-01-01

    Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few technolo......Next-generation sequencing has dramatically increased genome-wide profiling options and conceptually initiates the possibility for personalized cancer therapy. State-of-the-art sequencing studies yield large candidate gene sets comprising dozens or hundreds of mutated genes. However, few...... technologies are available for the systematic downstream evaluation of these results to identify novel starting points of future cancer therapies. We improved and extended a site-specific recombination-based system for systematic analysis of the individual functions of a large number of candidate genes....... This was facilitated by a novel system for the construction of isogenic constitutive and inducible gain- and loss-of-function cell lines. Additionally, we demonstrate the construction of isogenic cell lines with combinations of the traits for advanced functional in vitro analyses. In a proof-of-concept experiment...

  1. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

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    Carlos A Santiviago

    2009-07-01

    Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

  2. A high-throughput method to examine protein-nucleotide interactions identifies targets of the bacterial transcriptional regulatory protein fur.

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    Chunxiao Yu

    Full Text Available The Ferric uptake regulatory protein (Fur is a transcriptional regulatory protein that functions to control gene transcription in response to iron in a number of pathogenic bacteria. In this study, we applied a label-free, quantitative and high-throughput analysis method, Interferometric Reflectance Imaging Sensor (IRIS, to rapidly characterize Fur-DNA interactions in vitro with predicted Fur binding sequences in the genome of Neisseria gonorrhoeae, the causative agent of the sexually transmitted disease gonorrhea. IRIS can easily be applied to examine multiple protein-protein, protein-nucleotide and nucleotide-nucleotide complexes simultaneously and demonstrated here that seventy percent of the predicted Fur boxes in promoter regions of iron-induced genes bound to Fur in vitro with a range of affinities as observed using this microarray screening technology. Combining binding data with mRNA expression levels in a gonococcal fur mutant strain allowed us to identify five new gonococcal genes under Fur-mediated direct regulation.

  3. Analysis of pools of targeted Salmonella deletion mutants identifies novel genes affecting fitness during competitive infection in mice.

    Directory of Open Access Journals (Sweden)

    Carlos A Santiviago

    2009-07-01

    Full Text Available Pools of mutants of minimal complexity but maximal coverage of genes of interest facilitate screening for genes under selection in a particular environment. We constructed individual deletion mutants in 1,023 Salmonella enterica serovar Typhimurium genes, including almost all genes found in Salmonella but not in related genera. All mutations were confirmed simultaneously using a novel amplification strategy to produce labeled RNA from a T7 RNA polymerase promoter, introduced during the construction of each mutant, followed by hybridization of this labeled RNA to a Typhimurium genome tiling array. To demonstrate the ability to identify fitness phenotypes using our pool of mutants, the pool was subjected to selection by intraperitoneal injection into BALB/c mice and subsequent recovery from spleens. Changes in the representation of each mutant were monitored using T7 transcripts hybridized to a novel inexpensive minimal microarray. Among the top 120 statistically significant spleen colonization phenotypes, more than 40 were mutations in genes with no previously known role in this model. Fifteen phenotypes were tested using individual mutants in competitive assays of intraperitoneal infection in mice and eleven were confirmed, including the first two examples of attenuation for sRNA mutants in Salmonella. We refer to the method as Array-based analysis of cistrons under selection (ABACUS.

  4. PROBIT MODEL ANALYSIS OF SMALLHOLDER’S FARMERS DECISION TO USE AGROCHEMICAL INPUTS IN GWAGWALADA AND KUJE AREA COUNCILS OF FEDERAL CAPITAL TERRITORY, ABUJA, NIGERIA

    Directory of Open Access Journals (Sweden)

    Omotayo Olugbenga Alabi

    2014-01-01

    Full Text Available This study examined Probit model analysis of smallholder’s farmers decision to use agrochemical inputs in Gwagwalada and Kuje Area Councils of Federal Capital Territory, Abuja, Nigeria. Primary data were used for this study. Data were obtained using structured questionnaire. The questionnaires were administered to sixty smallholder’s farmers sampled using a two-stage sampling technique. Data obtained were analyzed using descriptive statistics and Probit model. Eight estimators, age; farm-size; education–level; extension services; access to credit; off-farm income; experiences in farming; in the Probit model were found statistically significant. Results show that the probability of using agrochemical inputs increases with age; farm-size; family-size; education-level; extension services; experiences in farming but decreases where they have off-farm income and access to credits. Mc Fadden Pseudo-R 2 gives 0.6866 and Probit model correctly classified 93%. This study concluded that capacity of agricultural extension agents needs to be improved in the study area to educate farmers to invest in agrochemicals and improved agricultural technologies. Also, Government needs to improve on good road networks and appropriate policies to regulate standard, use, safety needs and environment of use of agrochemicals in the study area.

  5. Employees’ Participation in Decision Making and the Hospitality Industry in Nigeria. An Investigative Study of Selected Hotels in the Federal Capital Territory, Abuja, Nigeria

    Directory of Open Access Journals (Sweden)

    Isichei Ejikeme Emmanuel

    2015-12-01

    Full Text Available The study investigated employee’s participation in decision making and the hospitality industry in Nigeria, a study of selected hotels in the federal capital territory in Abuja. To guide the study, research questions and hypotheses were designed. An in-depth review of related literature was carried out. Primary data method was used for the study and the instrument used was a questionnaire survey designed using the Likert scale questionnaire format and administered to sixty respondents. Test of reliability of research instrument showed a result of .874 using Cronbach Alpha. Content method was used for validity of the research instrument. Two Hypotheses was postulated in line with the objectives and research question to test the significance of the research problems. The study used linear correlation and regression for analysis of data obtained from research instrument, which proved the alternate hypothesis significant in the two hypotheses tested. Findings in the study showed that employee’s participation in decision making impacts on the performance of hotels in Nigeria. The study concludes there is a positive relationship between extent of employee’s participation in decision making and organisational performance. The study recommends among others that participation of employees’ should not just be partial but holistic to give them a sense of belonging.

  6. Proteomic analyses of the SMYD family interactomes identify HSP90 as a novel target for SMYD2

    Institute of Scientific and Technical Information of China (English)

    Mohamed Abu-Farha; Sylvain Lanouette; Fred Elisma; Véronique Tremblay; Jeffery Butson; Daniel Figeys; Jean-Francois Couture

    2011-01-01

    The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes,including gene expression regulation and DNA damage response.Initially identified as genuine histone methyltransferases,specific members of this family have recently been shown to methylate non-histone proteins such as p53,VEGFR,and the retinoblastoma tumor suppressor (pRb).To gain further functional insights into this family of KMTs,we generated the protein interaction network for three different human SMYD proteins (SMYD2,SMYD3,and SMYDS).Characterization of each SMYD protein network revealed that they associate with both shared and unique sets of proteins.Among those,we found that HsP90 and several of its co-chaperones interact specifically with the tetratrico peptide repeat (TPR)-containing SMYD2 and SMYD3.Moreover,using proteomic and biochemical techniques,we provide evidence that SMYD2 methylates K209 and K615 on HSP90 nucleotide-binding and dimerization domains,respectively.In addition,we found that each methylation site displays unique reactivity in regard to the presence of HsP90 co-chaperones,pH,and demethylation by the lysine amine oxidase LSD1,suggesting that alternative mechanisms control HsP90 methylation by SMYD2.Altogether,this study highlights the ability of SMYD proteins to form unique protein complexes that may underlie their various biological functions and the SMYD2-mediated methylation of the key molecular chaperone HSP90.%The SMYD (SET and MYND domain) family of lysine methyltransferases (KMTs) plays pivotal roles in various cellular processes, including gene expression regulation and DNA damage response. Initially identified as genuine histone methyltransferases, specific members of this family have recently been shown to methylate non-histone proteins such as p53, VEGFR, and the retinoblastoma tumor suppressor (pRb). To gain further functional insights into this family of KMTs, we generated the protein interaction

  7. Identifying high risk individuals for targeted lung cancer screening: Independent validation of the PLCOm2012 risk prediction tool.

    Science.gov (United States)

    Weber, Marianne; Yap, Sarsha; Goldsbury, David; Manners, David; Tammemagi, Martin; Marshall, Henry; Brims, Fraser; McWilliams, Annette; Fong, Kwun; Kang, Yoon Jung; Caruana, Michael; Banks, Emily; Canfell, Karen

    2017-07-15

    Lung cancer screening with computerised tomography holds promise, but optimising the balance of benefits and harms via selection of a high risk population is critical. PLCOm2012 is a logistic regression model based on U.S. data, incorporating sociodemographic and health factors, which predicts 6-year lung cancer risk among ever-smokers, and thus may better predict those who might benefit from screening than criteria based solely on age and smoking history. We aimed to validate the performance of PLCOm2012 in predicting lung cancer outcomes in a cohort of Australian smokers. Predicted risk of lung cancer was calculated using PLCOm2012 applied to baseline data from 95,882 ever-smokers aged ≥45 years in the 45 and Up Study (2006-2009). Predictions were compared to lung cancer outcomes captured to June 2014 via linkage to population-wide health databases; a total of 1,035 subsequent lung cancer diagnoses were identified. PLCOm2012 had good discrimination (area under the receiver-operating-characteristic-curve; AUC 0.80, 95%CI 0.78-0.81) and excellent calibration (mean and 90th percentiles of absolute risk difference between observed and predicted outcomes: 0.006 and 0.016, respectively). Sensitivity (69.4%, 95%CI, 65.6-73.0%) of the PLCOm2012 criteria in the 55-74 year age group for predicting lung cancers was greater than that using criteria based on ≥30 pack-years smoking and ≤15 years quit (57.3%, 53.3-61.3%; p cancer screening using PLCOm2012 might improve the balance of benefits versus harms, and cost-effectiveness of lung cancer screening. © 2017 UICC.

  8. New biomarkers of coffee consumption identified by the non-targeted metabolomic profiling of cohort study subjects.

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    Joseph A Rothwell

    Full Text Available Coffee contains various bioactives implicated with human health and disease risk. To accurately assess the effects of overall consumption upon health and disease, individual intake must be measured in large epidemiological studies. Metabolomics has emerged as a powerful approach to discover biomarkers of intake for a large range of foods. Here we report the profiling of the urinary metabolome of cohort study subjects to search for new biomarkers of coffee intake. Using repeated 24-hour dietary records and a food frequency questionnaire, 20 high coffee consumers (183-540 mL/d and 19 low consumers were selected from the French SU.VI.MAX2 cohort. Morning spot urine samples from each subject were profiled by high-resolution mass spectrometry. Partial least-square discriminant analysis of multidimensional liquid chromatography-mass spectrometry data clearly distinguished high consumers from low via 132 significant (p-value<0.05 discriminating features. Ion clusters whose intensities were most elevated in the high consumers were annotated using online and in-house databases and their identities checked using commercial standards and MS-MS fragmentation. The best discriminants, and thus potential markers of coffee consumption, were the glucuronide of the diterpenoid atractyligenin, the diketopiperazine cyclo(isoleucyl-prolyl, and the alkaloid trigonelline. Some caffeine metabolites, such as 1-methylxanthine, were also among the discriminants, however caffeine may be consumed from other sources and its metabolism is subject to inter-individual variation. Receiver operating characteristics curve analysis showed that the biomarkers identified could be used effectively in combination for increased sensitivity and specificity. Once validated in other cohorts or intervention studies, these specific single or combined biomarkers will become a valuable alternative to assessment of coffee intake by dietary survey and finally lead to a better understanding of

  9. A network biology approach evaluating the anticancer effects of bortezomib identifies SPARC as a therapeutic target in adult T-cell leukemia cells

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    Yu Zhang

    2008-10-01

    Full Text Available Junko H Ohyashiki1, Ryoko Hamamura2, Chiaki Kobayashi2, Yu Zhang2, Kazuma Ohyashiki21Intractable Immune System Disease Research Center, Tokyo Medical University, Tokyo, Japan; 2First Department of Internal Medicine, Tokyo Medical University, Tokyo, JapanAbstract: There is a need to identify the regulatory gene interaction of anticancer drugs on target cancer cells. Whole genome expression profiling offers promise in this regard, but can be complicated by the challenge of identifying the genes affected by hundreds to thousands of genes that induce changes in expression. A proteasome inhibitor, bortezomib, could be a potential therapeutic agent in treating adult T-cell leukemia (ATL patients, however, the underlying mechanism by which bortezomib induces cell death in ATL cells via gene regulatory network has not been fully elucidated. Here we show that a Bayesian statistical framework by VoyaGene® identified a secreted protein acidic and rich in cysteine (SPARC gene, a tumor-invasiveness related gene, as a possible modulator of bortezomib-induced cell death in ATL cells. Functional analysis using RNAi experiments revealed that inhibition of the expression SPARC by siRNA enhanced the apoptotic effect of bortezomib on ATL cells in accordance with an increase of cleaved caspase 3. Targeting SPARC may help to treat ATL patients in combination with bortezomib. This work shows that a network biology approach can be used advantageously to identify the genetic interaction related to anticancer effects.Keywords: network biology, adult T cell leukemia, bortezomib, SPARC

  10. Substrate profiling of human vaccinia-related kinases identifies coilin, a Cajal body nuclear protein, as a phosphorylation target with neurological implications.

    Science.gov (United States)

    Sanz-García, Marta; Vázquez-Cedeira, Marta; Kellerman, Efrat; Renbaum, Paul; Levy-Lahad, Ephrat; Lazo, Pedro A

    2011-12-21

    Protein phosphorylation by kinases plays a central role in the regulation and coordination of multiple biological processes. In general, knowledge on kinase specificity is restricted to substrates identified in the context of specific cellular responses, but kinases are likely to have multiple additional substrates and be integrated in signaling networks that might be spatially and temporally different, and in which protein complexes and subcellular localization can play an important role. In this report the substrate specificity of atypical human vaccinia-related kinases (VRK1 and VRK2) using a human peptide-array containing 1080 sequences phosphorylated in known signaling pathways has been studied. The two kinases identify a subset of potential peptide targets, all of them result in a consensus sequence composed of at least four basic residues in peptide targets. Linear peptide arrays are therefore a useful approach in the characterization of kinases and substrate identification, which can contribute to delineate the signaling network in which VRK proteins participate. One of these target proteins is coilin; a basic protein located in nuclear Cajal bodies. Coilin is phosphorylated in Ser184 by both VRK1 and VRK2. Coilin colocalizes and interacts with VRK1 in Cajal bodies, but not with the mutant VRK1 (R358X). VRK1 (R358X) is less active than VRK1. Altered regulation of coilin might be implicated in several neurological diseases such as ataxias and spinal muscular atrophies.

  11. Integration analysis of quantitative proteomics and transcriptomics data identifies potential targets of frizzled-8 protein-related antiproliferative factor in vivo.

    Science.gov (United States)

    Yang, Wei; Kim, Yongsoo; Kim, Taek-Kyun; Keay, Susan K; Kim, Kwang Pyo; Steen, Hanno; Freeman, Michael R; Hwang, Daehee; Kim, Jayoung

    2012-12-01

    What's known on the subject? and What does the study add? Interstitial cystitis (IC) is a prevalent and debilitating pelvic disorder generally accompanied by chronic pain combined with chronic urinating problems. Over one million Americans are affected, especially middle-aged women. However, its aetiology or mechanism remains unclear. No efficient drug has been provided to patients. Several urinary biomarker candidates have been identified for IC; among the most promising is antiproliferative factor (APF), whose biological activity is detectable in urine specimens from >94% of patients with both ulcerative and non-ulcerative IC. The present study identified several important mediators of the effect of APF on bladder cell physiology, suggesting several candidate drug targets against IC. In an attempt to identify potential proteins and genes regulated by APF in vivo, and to possibly expand the APF-regulated network identified by stable isotope labelling by amino acids in cell culture (SILAC), we performed an integration analysis of our own SILAC data and the microarray data of Gamper et al. (2009) BMC Genomics 10: 199. Notably, two of the proteins (i.e. MAPKSP1 and GSPT1) that are down-regulated by APF are involved in the activation of mTORC1, suggesting that the mammalian target of rapamycin (mTOR) pathway is potentially a critical pathway regulated by APF in vivo. Several components of the mTOR pathway are currently being studied as potential therapeutic targets in other diseases. Our analysis suggests that this pathway might also be relevant in the design of diagnostic tools and medications targeting IC. • To enhance our understanding of the interstitial cystitis urine biomarker antiproliferative factor (APF), as well as interstitial cystitis biology more generally at the systems level, we reanalyzed recently published large-scale quantitative proteomics and in vivo transcriptomics data sets using an integration analysis tool that we have developed. • To

  12. Increasing organizational energy conservation behaviors: Comparing the theory of planned behavior and reasons theory for identifying specific motivational factors to target for change

    Science.gov (United States)

    Finlinson, Scott Michael

    Social scientists frequently assess factors thought to underlie behavior for the purpose of designing behavioral change interventions. Researchers commonly identify these factors by examining relationships between specific variables and the focal behaviors being investigated. Variables with the strongest relationships to the focal behavior are then assumed to be the most influential determinants of that behavior, and therefore often become the targets for change in a behavioral change intervention. In the current proposal, multiple methods are used to compare the effectiveness of two theoretical frameworks for identifying influential motivational factors. Assessing the relative influence of all factors and sets of factors for driving behavior should clarify which framework and methodology is the most promising for identifying effective change targets. Results indicated each methodology adequately predicted the three focal behaviors examined. However, the reasons theory approach was superior for predicting factor influence ratings compared to the TpB approach. While common method variance contamination had minimal impact on the results or conclusions derived from the present study's findings, there were substantial differences in conclusions depending on the questionnaire design used to collect the data. Examples of applied uses of the present study are discussed.

  13. System-based proteomic and metabonomic analysis of the Df(16)A+/− mouse identifies potential miR-185 targets and molecular pathway alterations

    Science.gov (United States)

    Wesseling, H; Xu, B; Want, E J; Holmes, E; Guest, P C; Karayiorgou, M; Gogos, J A; Bahn, S

    2017-01-01

    Deletions on chromosome 22q11.2 are a strong genetic risk factor for development of schizophrenia and cognitive dysfunction. We employed shotgun liquid chromatography–mass spectrometry (LC-MS) proteomic and metabonomic profiling approaches on prefrontal cortex (PFC) and hippocampal (HPC) tissue from Df(16)A+/− mice, a model of the 22q11.2 deletion syndrome. Proteomic results were compared with previous transcriptomic profiling studies of the same brain regions. The aim was to investigate how the combined effect of the 22q11.2 deletion and the corresponding miRNA dysregulation affects the cell biology at the systems level. The proteomic brain profiling analysis revealed PFC and HPC changes in various molecular pathways associated with chromatin remodelling and RNA transcription, indicative of an epigenetic component of the 22q11.2DS. Further, alterations in glycolysis/gluconeogenesis, mitochondrial function and lipid biosynthesis were identified. Metabonomic profiling substantiated the proteomic findings by identifying changes in 22q11.2 deletion syndrome (22q11.2DS)-related pathways, such as changes in ceramide phosphoethanolamines, sphingomyelin, carnitines, tyrosine derivates and panthothenic acid. The proteomic findings were confirmed using selected reaction monitoring mass spectrometry, validating decreased levels of several proteins encoded on 22q11.2, increased levels of the computationally predicted putative miR-185 targets UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase 110 kDa subunit (OGT1) and kinesin heavy chain isoform 5A and alterations in the non-miR-185 targets serine/threonine-protein phosphatase 2B catalytic subunit gamma isoform, neurofilament light chain and vesicular glutamate transporter 1. Furthermore, alterations in the proteins associated with mammalian target of rapamycin signalling were detected in the PFC and with glutamatergic signalling in the hippocampus. Based on the proteomic and metabonomic findings, we were

  14. A systems biology approach identified different regulatory networks targeted by KSHV miR-K12-11 in B cells and endothelial cells.

    Science.gov (United States)

    Yang, Yajie; Boss, Isaac W; McIntyre, Lauren M; Renne, Rolf

    2014-08-08

    Kaposi's sarcoma associated herpes virus (KSHV) is associated with tumors of endothelial and lymphoid origin. During latent infection, KSHV expresses miR-K12-11, an ortholog of the human tumor gene hsa-miR-155. Both gene products are microRNAs (miRNAs), which are important post-transcriptional regulators that contribute to tissue specific gene expression. Advances in target identification technologies and molecular interaction databases have allowed a systems biology approach to unravel the gene regulatory networks (GRNs) triggered by miR-K12-11 in endothelial and lymphoid cells. Understanding the tissue specific function of miR-K12-11 will help to elucidate underlying mechanisms of KSHV pathogenesis. Ectopic expression of miR-K12-11 differentially affected gene expression in BJAB cells of lymphoid origin and TIVE cells of endothelial origin. Direct miRNA targeting accounted for a small fraction of the observed transcriptome changes: only 29 genes were identified as putative direct targets of miR-K12-11 in both cell types. However, a number of commonly affected biological pathways, such as carbohydrate metabolism and interferon response related signaling, were revealed by gene ontology analysis. Integration of transcriptome profiling, bioinformatic algorithms, and databases of protein-protein interactome from the ENCODE project identified different nodes of GRNs utilized by miR-K12-11 in a tissue-specific fashion. These effector genes, including cancer associated transcription factors and signaling proteins, amplified the regulatory potential of a single miRNA, from a small set of putative direct targets to a larger set of genes. This is the first comparative analysis of miRNA-K12-11's effects in endothelial and B cells, from tissues infected with KSHV in vivo. MiR-K12-11 was able to broadly modulate gene expression in both cell types. Using a systems biology approach, we inferred that miR-K12-11 establishes its GRN by both repressing master TFs and influencing

  15. Identifying Targets from Filtering Effects

    Science.gov (United States)

    2012-10-24

    I. Abarbanel, R. Brown., J. J. Sidorowich, and L. S. Tsmring, "The Analysis of Observed Chaotic Data in Physical Systems," Reviews of Modern ...A. Taflove and S. C. Hagness, Computational Electrodynamics : The Finite Difference Time Domain Method. Norwood, MA: Artech House, 2005.

  16. MicroRNA-Target Network Inference and Local Network Enrichment Analysis Identify Two microRNA Clusters with Distinct Functions in Head and Neck Squamous Cell Carcinoma

    Directory of Open Access Journals (Sweden)

    Steffen Sass

    2015-12-01

    Full Text Available MicroRNAs represent ~22 nt long endogenous small RNA molecules that have been experimentally shown to regulate gene expression post-transcriptionally. One main interest in miRNA research is the investigation of their functional roles, which can typically be accomplished by identification of mi-/mRNA interactions and functional annotation of target gene sets. We here present a novel method “miRlastic”, which infers miRNA-target interactions using transcriptomic data as well as prior knowledge and performs functional annotation of target genes by exploiting the local structure of the inferred network. For the network inference, we applied linear regression modeling with elastic net regularization on matched microRNA and messenger RNA expression profiling data to perform feature selection on prior knowledge from sequence-based target prediction resources. The novelty of miRlastic inference originates in predicting data-driven intra-transcriptome regulatory relationships through feature selection. With synthetic data, we showed that miRlastic outperformed commonly used methods and was suitable even for low sample sizes. To gain insight into the functional role of miRNAs and to determine joint functional properties of miRNA clusters, we introduced a local enrichment analysis procedure. The principle of this procedure lies in identifying regions of high functional similarity by evaluating the shortest paths between genes in the network. We can finally assign functional roles to the miRNAs by taking their regulatory relationships into account. We thoroughly evaluated miRlastic on a cohort of head and neck cancer (HNSCC patients provided by The Cancer Genome Atlas. We inferred an mi-/mRNA regulatory network for human papilloma virus (HPV-associated miRNAs in HNSCC. The resulting network best enriched for experimentally validated miRNA-target interaction, when compared to common methods. Finally, the local enrichment step identified two functional

  17. Targeted exome sequencing identifies novel compound heterozygous mutations in P3H1 in a fetus with osteogenesis imperfecta type VIII.

    Science.gov (United States)

    Huang, Yanru; Mei, Libin; Lv, Weigang; Li, Haoxian; Zhang, Rui; Pan, Qian; Tan, Hu; Guo, Jing; Luo, Xiaomei; Chen, Chen; Liang, Desheng; Wu, Lingqian

    2017-01-01

    Osteogenesis imperfecta (OI) is a highly clinically and genetically heterogeneous group of disorders. It is difficult to identify severe OI in the perinatal period. Here, a Chinese woman with a suspected history of fetal OI was referred to our institution at 19weeks of gestation, due to ultrasound inspection during antenatal screening, which revealed bulbous metaphyses, short humeri, and short thick bent femora in the fetus. Using targeted exome sequencing of 248 genes known to be involved in skeletal system diseases, we identified novel compound heterozygous mutation in the P3H1 gene in the fetus with OI type VIII: c.105_120del (p.D36Rfs*16) and c.2164C>T (p.Q722*). These two mutations were inherited from the father and mother, respectively. The mRNA level of P3H1 wasn't changed suggested that mRNA with this mutation escaped from nonsense-mediated RNA decay. Besides, the level of P3H1 was absence while the CRTAP was mildly decreased. In conclusion, our findings imply this novel compound heterozygous mutation as the molecular pathogenetic in a Chinese fetus with OI type VIII, and demonstrate that targeted next-generation sequencing (NGS) is an accurate, rapid, and cost-effective method in the genetic diagnosis of fetal skeletal dysplasia with genetic and clinical heterogeneity, especially for autosomal recessive skeletal disorders.

  18. Deep Sequencing Identifies Tissue-Specific MicroRNAs and Their Target Genes Involving in the Biosynthesis of Tanshinones in Salvia miltiorrhiza

    Science.gov (United States)

    Xu, Xiangbin; Jiang, Qinghua; Ma, Xiuyan; Ying, Qicai; Shen, Bo; Qian, Yongsheng; Song, Hongmiao; Wang, Huizhong

    2014-01-01

    Salvia miltiorrhiza is one of the most popular traditional medicinal herbs in Asian nations. Its dried root contains a number of tanshinones, protocatechuic aldehyde, salvianolic acid B and rosmarinic, and is used for the treatment of various diseases. The finding of microRNAs (miRNAs) and their target genes will help understand their biological role on the biosynthesis of tanshinones in S. miltiorrhiza. In the present study, a total of 452 known miRNAs corresponding to 589 precursor miRNAs (pre-miRNAs), and 40 novel miRNAs corresponding to 24 pre-miRNAs were identified in different tissues of S. miltiorrhiza by high-throughput sequencing, respectively. Among them, 62 miRNAs express only in root, 95 miRNAs express only in stem, 19 miRNAs express only in leaf, and 71 miRNAs express only in flower, respectively. By the degradome analysis, 69 targets potentially cleaved by 25 miRNAs were identified. Among them, acetyl-CoA C-acetyltransferase was cleaved by miR5072, and involved in the biosynthesis of tanshinones. This study provided valuable information for understanding the tissue-specific expression patterns of miRNAs in S. miltiorrhiza, and offered a foundation for future studies of the miRNA-mediated biosynthesis of tanshinones. PMID:25365305

  19. Deep sequencing identifies tissue-specific microRNAs and their target genes involving in the biosynthesis of tanshinones in Salvia miltiorrhiza.

    Directory of Open Access Journals (Sweden)

    Xiangbin Xu

    Full Text Available Salvia miltiorrhiza is one of the most popular traditional medicinal herbs in Asian nations. Its dried root contains a number of tanshinones, protocatechuic aldehyde, salvianolic acid B and rosmarinic, and is used for the treatment of various diseases. The finding of microRNAs (miRNAs and their target genes will help understand their biological role on the biosynthesis of tanshinones in S. miltiorrhiza. In the present study, a total of 452 known miRNAs corresponding to 589 precursor miRNAs (pre-miRNAs, and 40 novel miRNAs corresponding to 24 pre-miRNAs were identified in different tissues of S. miltiorrhiza by high-throughput sequencing, respectively. Among them, 62 miRNAs express only in root, 95 miRNAs express only in stem, 19 miRNAs express only in leaf, and 71 miRNAs express only in flower, respectively. By the degradome analysis, 69 targets potentially cleaved by 25 miRNAs were identified. Among them, acetyl-CoA C-acetyltransferase was cleaved by miR5072, and involved in the biosynthesis of tanshinones. This study provided valuable information for understanding the tissue-specific expression patterns of miRNAs in S. miltiorrhiza, and offered a foundation for future studies of the miRNA-mediated biosynthesis of tanshinones.

  20. PDK1/Akt/PDE4D axis identified as a target for asthma remedy synergistic with β2 AR agonists by a natural agent arctigenin.

    Science.gov (United States)

    Fang, R; Cui, Q; Sun, J; Duan, X; Ma, X; Wang, W; Cheng, B; Liu, Y; Hou, Y; Bai, G

    2015-12-01

    Asthma is a heterogenetic disorder characterized by chronic inflammation with variable airflow obstruction and airway hyper-responsiveness. As the most potent and popular bronchodilators, β2 adrenergic receptor (β2 AR) agonists bind to the β2 ARs that are coupled via a stimulatory G protein to adenylyl cyclase, thereby improving cAMP accumulation and resulting in airway smooth muscle relaxation. We previously demonstrated arctigenin had a synergistic function with the β2 AR agonist, but the target for this remained elusive. Chemical proteomics capturing was used to enrich and uncover the target of arctigenin in human bronchial smooth muscle cells, and reverse docking and molecular dynamic stimulation were performed to evaluate the binding of arctigenin and its target. In vitro enzyme activities and protein levels were demonstrated with special kits and Western blotting. Finally, guinea pig tracheal muscle segregation and ex vivo function were analysed. Arctigenin bound to PDK1 with an ideal binding free energy -25.45 kcal/mol and inhibited PDK1 kinase activity without changing its protein level. Additionally, arctigenin reduced PKB/Akt-induced phosphorylation of PDE4D, which was first identified in this study. Attenuation of PDE4D resulted in cAMP accumulation in human bronchial smooth muscle. The inhibition of PDK1 showed a synergistic function with β2 AR agonists and relaxed the constriction of segregated guinea pig tracheal muscle. The PDK1/Akt/PDE4D axis serves as a novel asthma target, which may benefit airflow obstruction. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  1. Identifying Patient-Specific Epstein-Barr Nuclear Antigen-1 Genetic Variation and Potential Autoreactive Targets Relevant to Multiple Sclerosis Pathogenesis.

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    Monika Tschochner

    Full Text Available Epstein-Barr virus (EBV infection represents a major environmental risk factor for multiple sclerosis (MS, with evidence of selective expansion of Epstein-Barr Nuclear Antigen-1 (EBNA1-specific CD4+ T cells that cross-recognize MS-associated myelin antigens in MS patients. HLA-DRB1*15-restricted antigen presentation also appears to determine susceptibility given its role as a dominant risk allele. In this study, we have utilised standard and next-generation sequencing techniques to investigate EBNA-1 sequence variation and its relationship to HLA-DR15 binding affinity, as well as examining potential cross-reactive immune targets within the central nervous system proteome.Sanger sequencing was performed on DNA isolated from peripheral blood samples from 73 Western Australian MS cases, without requirement for primary culture, with additional FLX 454 Roche sequencing in 23 samples to identify low-frequency variants. Patient-derived viral sequences were used to predict HLA-DRB1*1501 epitopes (NetMHCII, NetMHCIIpan and candidates were evaluated for cross recognition with human brain proteins.EBNA-1 sequence variation was limited, with no evidence of multiple viral strains and only low levels of variation identified by FLX technology (8.3% nucleotide positions at a 1% cut-off. In silico epitope mapping revealed two known HLA-DRB1*1501-restricted epitopes ('AEG': aa 481-496 and 'MVF': aa 562-577, and two putative epitopes between positions 502-543. We identified potential cross-reactive targets involving a number of major myelin antigens including experimentally confirmed HLA-DRB1*15-restricted epitopes as well as novel candidate antigens within myelin and paranodal assembly proteins that may be relevant to MS pathogenesis.This study demonstrates the feasibility of obtaining autologous EBNA-1 sequences directly from buffy coat samples, and confirms divergence of these sequences from standard laboratory strains. This approach has identified a number of

  2. Environmental Modeling, The Natural Filter Buffer Targeting layers identify riparian forest and grass buffer opportunities by county. Land use and hydrology characteristics were used to identify potential riparian buffer locations., Published in 2014, Smaller than 1:100000 scale, Maryland Department of Natural Resources (DNR).

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    NSGIC Education | GIS Inventory — Environmental Modeling dataset current as of 2014. The Natural Filter Buffer Targeting layers identify riparian forest and grass buffer opportunities by county. Land...

  3. Lampe1: an ENU-germline mutation causing spontaneous hepatosteatosis identified through targeted exon-enrichment and next-generation sequencing.

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    Rachel Sheridan

    Full Text Available Using a small scale ENU mutagenesis approach we identified a recessive germline mutant, designated Lampe1 that exhibited growth retardation and spontaneous hepatosteatosis. Low resolution mapping based on 20 intercrossed Lampe1 mice revealed linkage to a ∼14 Mb interval on the distal site of chromosome 11 containing a total of 285 genes. Exons and 50 bp flanking sequences within the critical region were enriched with sequence capture microarrays and subsequently analyzed by next-generation sequencing. Using this approach 98.1 percent of the targeted DNA was covered with a depth of 10 or more reads per nucleotide and 3 homozygote mutations were identified. Two mutations represented intronic nucleotide changes whereas one mutation affected a splice donor site in intron 11-12 of Palmitoyl Acetyl-coenzyme A oxygenase-1 (Acox1, causing skipping of exon 12. Phenotyping of Acox1(Lampe1 mutants revealed a progression from hepatosteatosis to steatohepatitis, and ultimately hepatocellular carcinoma. The current approach provides a highly efficient and affordable method to identify causative mutations induced by ENU mutagenesis and animal models relevant to human pathology.

  4. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

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    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  5. Haploid genetic screens identify an essential role for PLP2 in the downregulation of novel plasma membrane targets by viral E3 ubiquitin ligases.

    Directory of Open Access Journals (Sweden)

    Richard T Timms

    Full Text Available The Kaposi's sarcoma-associated herpesvirus gene products K3 and K5 are viral ubiquitin E3 ligases which downregulate MHC-I and additional cell surface immunoreceptors. To identify novel cellular genes required for K5 function we performed a forward genetic screen in near-haploid human KBM7 cells. The screen identified proteolipid protein 2 (PLP2, a MARVEL domain protein of unknown function, as essential for K5 activity. Genetic loss of PLP2 traps the viral ligase in the endoplasmic reticulum, where it is unable to ubiquitinate and degrade its substrates. Subsequent analysis of the plasma membrane proteome of K5-expressing KBM7 cells in the presence and absence of PLP2 revealed a wide range of novel K5 targets, all of which required PLP2 for their K5-mediated downregulation. This work ascribes a critical function to PLP2 for viral ligase activity and underlines the power of non-lethal haploid genetic screens in human cells to identify the genes involved in pathogen manipulation of the host immune system.

  6. Hombres Sanos: exposure and response to a social marketing HIV prevention campaign targeting heterosexually identified Latino men who have sex with men and women.

    Science.gov (United States)

    Martínez-Donate, Ana P; Zellner, Jennifer A; Fernández-Cerdeño, Araceli; Sañudo, Fernando; Hovell, Melbourne F; Sipan, Carol L; Engelberg, Moshe; Ji, Ming

    2009-10-01

    This study examined the reach and impact of a social marketing intervention to reduce HIV risk among heterosexually identified (HI) Latino men who have sex with men and women (MSMW). Repeated cross-sectional intercept surveys were conducted in selected community venues during and after the campaign with 1,137 HI Latino men. Of them, 6% were classified as HI Latino MSMW. On average, 85.9% of the heterosexual respondents and 86.8% of the HI MSMW subsample reported exposure to the campaign. Responses to the campaign included having made an appointment for a male health exam that included HIV testing and using condoms. Campaign exposure was significantly associated with HIV testing behavior and intentions and with knowledge of where to get tested. The campaign reached its underserved target audience and stimulated preventive behaviors. Social marketing represents a promising approach for HIV prevention among HI Latinos, in general, and HI Latino MSMW, in particular.

  7. Bioinformatic analysis of patient-derived ASPS gene expressions and ASPL-TFE3 fusion transcript levels identify potential therapeutic targets.

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    David G Covell

    Full Text Available Gene expression data, collected from ASPS tumors of seven different patients and from one immortalized ASPS cell line (ASPS-1, was analyzed jointly with patient ASPL-TFE3 (t(X;17(p11;q25 fusion transcript data to identify disease-specific pathways and their component genes. Data analysis of the pooled patient and ASPS-1 gene expression data, using conventional clustering methods, revealed a relatively small set of pathways and genes characterizing the biology of ASPS. These results could be largely recapitulated using only the gene expression data collected from patient tumor samples. The concordance between expression measures derived from ASPS-1 and both pooled and individual patient tumor data provided a rationale for extending the analysis to include patient ASPL-TFE3 fusion transcript data. A novel linear model was exploited to link gene expressions to fusion transcript data and used to identify a small set of ASPS-specific pathways and their gene expression. Cellular pathways that appear aberrantly regulated in response to the t(X;17(p11;q25 translocation include the cell cycle and cell adhesion. The identification of pathways and gene subsets characteristic of ASPS support current therapeutic strategies that target the FLT1 and MET, while also proposing additional targeting of genes found in pathways involved in the cell cycle (CHK1, cell adhesion (ARHGD1A, cell division (CDC6, control of meiosis (RAD51L3 and mitosis (BIRC5, and chemokine-related protein tyrosine kinase activity (CCL4.

  8. Transcriptome Profiling Identifies Multiplexin as a Target of SAGA Deubiquitinase Activity in Glia Required for Precise Axon Guidance During Drosophila Visual Development

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    Jingqun Ma

    2016-08-01

    Full Text Available The Spt-Ada-Gcn5 Acetyltransferase (SAGA complex is a transcriptional coactivator with histone acetylase and deubiquitinase activities that plays an important role in visual development and function. In Drosophila melanogaster, four SAGA subunits are required for the deubiquitination of monoubiquitinated histone H2B (ubH2B: Nonstop, Sgf11, E(y2, and Ataxin 7. Mutations that disrupt SAGA deubiquitinase activity cause defects in neuronal connectivity in the developing Drosophila visual system. In addition, mutations in SAGA result in the human progressive visual disorder spinocerebellar ataxia type 7 (SCA7. Glial cells play a crucial role in both the neuronal connectivity defect in nonstop and sgf11 flies, and in the retinal degeneration observed in SCA7 patients. Thus, we sought to identify the gene targets of SAGA deubiquitinase activity in glia in the Drosophila larval central nervous system. To do this, we enriched glia from wild-type, nonstop, and sgf11 larval optic lobes using affinity-purification of KASH-GFP tagged nuclei, and then examined each transcriptome using RNA-seq. Our analysis showed that SAGA deubiquitinase activity is required for proper expression of 16% of actively transcribed genes in glia, especially genes involved in proteasome function, protein folding and axon guidance. We further show that the SAGA deubiquitinase-activated gene Multiplexin (Mp is required in glia for proper photoreceptor axon targeting. Mutations in the human ortholog of Mp, COL18A1, have been identified in a family with a SCA7-like progressive visual disorder, suggesting that defects in the expression of this gene in SCA7 patients could play a role in the retinal degeneration that is unique to this ataxia.

  9. Clinical Genotyping of Non–Small Cell Lung Cancers Using Targeted Next-Generation Sequencing: Utility of Identifying Rare and Co-mutations in Oncogenic Driver Genes

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    Laura J. Tafe

    2016-09-01

    Full Text Available Detection of somatic mutations in non–small cell lung cancers (NSCLCs, especially adenocarcinomas, is important for directing patient care when targeted therapy is available. Here, we present our experience with genotyping NSCLC using the Ion Torrent Personal Genome Machine (PGM and the AmpliSeq Cancer Hotspot Panel v2. We tested 453 NSCLC samples from 407 individual patients using the 50 gene AmpliSeq Cancer Hotspot Panel v2 from May 2013 to July 2015. Using 10 ng of DNA, up to 11 samples were simultaneously sequenced on the Ion Torrent PGM (316 and 318 chips. We identified variants with the Ion Torrent Variant Caller Plugin, and Golden Helix's SVS software was used for annotation and prediction of the significance of the variants. Three hundred ninety-eight samples were successfully sequenced (12.1% failure rate. In all, 633 variants in 41 genes were detected with a median of 2 (range of 0 to 7 variants per sample. Mutations detected in BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA were considered potentially actionable and were identified in 237 samples, most commonly in KRAS (37.9%, EGFR (11.1%, BRAF (4.8%, and PIK3CA (4.3%. In our patient population, all mutations in EGFR, KRAS, and BRAF were mutually exclusive. The Ion Torrent Ampliseq technology can be utilized on small biopsy and cytology specimens, requires very little input DNA, and can be applied in clinical laboratories for genotyping of NSCLC. This targeted next-generation sequencing approach allows for detection of common and also rare mutations that are clinically actionable in multiple patients simultaneously.

  10. Targeting the Dbl and dock-family RhoGEFs: a yeast-based assay to identify cell-active inhibitors of Rho-controlled pathways.

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    Blangy, Anne; Fort, Philippe

    2013-01-01

    The Ras-like superfamily of low molecular weight GTPases is made of five major families (Arf/Sar, Rab, Ran, Ras, and Rho), highly conserved across evolution. This is in keeping with their roles in basic cellular functions (endo/exocytosis, vesicular trafficking, nucleocytoplasmic trafficking, cell signaling, proliferation and apoptosis, gene regulation, F-actin dynamics), whose alterations are associated with various types of diseases, in particular cancer, neurodegenerative, cardiovascular, and infectious diseases. For these reasons, Ras-like pathways are of great potential in therapeutics and identifying inhibitors that decrease signaling activity is under intense research. Along this line, guanine exchange factors (GEFs) represent attractive targets. GEFs are proteins that promote the active GTP-bound state of GTPases and represent the major entry points whereby extracellular cues are converted into Ras-like signaling. We previously developed the yeast exchange assay (YEA), an experimental setup in the yeast in which activity of a mammalian GEF can be monitored by auxotrophy and color reporter genes. This assay was further engineered for medium-throughput screening of GEF inhibitors, which can readily select for cell-active and specific compounds. We report here on the successful identification of inhibitors against Dbl and CZH/DOCK-family members, GEFs for Rho GTPases, and on the experimental setup to screen for inhibitors of GEFs of the Arf family. We also discuss on inhibitors developed using virtual screening (VS), which target the GEF/GTPase interface with high efficacy and specificity. We propose that using VS and YEA in combination may represent a method of choice for identifying specific and cell-active GEF inhibitors. © 2013 Elsevier Inc. All rights reserved.

  11. MirSNP, a database of polymorphisms altering miRNA target sites, identifies miRNA-related SNPs in GWAS SNPs and eQTLs

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    Liu Chenxing

    2012-11-01

    Full Text Available Abstract Background Numerous single nucleotide polymorphisms (SNPs associated with complex diseases have been identified by genome-wide association studies (GWAS and expression quantitative trait loci (eQTLs studies. However, few of these SNPs have explicit biological functions. Recent studies indicated that the SNPs within the 3’UTR regions of susceptibility genes could affect complex traits/diseases by affecting the function of miRNAs. These 3’UTR SNPs are functional candidates and therefore of interest to GWAS and eQTL researchers. Description We developed a publicly available online database, MirSNP (http://cmbi.bjmu.edu.cn/mirsnp, which is a collection of human SNPs in predicted miRNA-mRNA binding sites. We identified 414,510 SNPs that might affect miRNA-mRNA binding. Annotations were added to these SNPs to predict whether a SNP within the target site would decrease/break or enhance/create an miRNA-mRNA binding site. By applying MirSNP database to three brain eQTL data sets, we identified four unreported SNPs (rs3087822, rs13042, rs1058381, and rs1058398, which might affect miRNA binding and thus affect the expression of their host genes in the brain. We also applied the MirSNP database to our GWAS for schizophrenia: seven predicted miRNA-related SNPs (p  Conclusion MirSNP could identify the putative miRNA-related SNPs from GWAS and eQTLs researches and provide the direction for subsequent functional researches.

  12. SPON2, a newly identified target gene of MACC1, drives colorectal cancer metastasis in mice and is prognostic for colorectal cancer patient survival.

    Science.gov (United States)

    Schmid, F; Wang, Q; Huska, M R; Andrade-Navarro, M A; Lemm, M; Fichtner, I; Dahlmann, M; Kobelt, D; Walther, W; Smith, J; Schlag, P M; Stein, U

    2016-11-17

    MACC1 (metastasis associated in colon cancer 1) is a prognostic biomarker for tumor progression, metastasis and survival of a variety of solid cancers including colorectal cancer (CRC). Here we aimed to identify the MACC1-induced transcriptome and key players mediating the MACC1-induced effects in CRC. We performed microarray analyses using CRC cells ectopically overexpressing MACC1. We identified more than 1300 genes at least twofold differentially expressed, including the gene SPON2 (Spondin 2) as 90-fold upregulated transcriptional target of MACC1. MACC1-dependent SPON2 expression regulation was validated on mRNA and protein levels in MACC1 high (endogenously or ectopically) and low (endogenously or by knockdown) expressing cells. Chromatin immunoprecipitation analysis demonstrated the binding of MACC1 to the gene promoter of SPON2. In cell culture, ectopic SPON2 overexpression induced cell viability, migration, invasion and colony formation in endogenously MACC1 and SPON2 low expressing cells, whereas SPON2 knockdown reduced proliferative, migratory and invasive abilities in CRC cells with high endogenous MACC1 and SPON2 expression. In intrasplenically transplanted NOD/SCID mice, metastasis induction was analyzed with control or SPON2-overexpressing CRC cells. Tumors with SPON2 overexpression induced liver metastasis (vs control animals without any metastases, P=0.0036). In CRC patients, SPON2 expression was determined in primary tumors (stages I-III), and survival time was analyzed by Kaplan-Meier method. CRC patients with high SPON2 expressing primary tumors demonstrated 8 months shorter metastasis-free survival (MFS) compared with patients with low SPON2 levels (P=0.053). Combining high levels of SPON2 and MACC1 improved the identification of high-risk patients with a 20-month shorter MFS vs patients with low biomarker expression. In summary, SPON2 is a transcriptional target of the metastasis gene MACC1. SPON2 induces cell motility in vitro and CRC

  13. RNA-Seq analysis of rye-grass transcriptomic response to an herbicide inhibiting acetolactate-synthase identifies transcripts linked to non-target-site-based resistance.

    Science.gov (United States)

    Duhoux, Arnaud; Carrère, Sébastien; Gouzy, Jérôme; Bonin, Ludovic; Délye, Christophe

    2015-03-01

    Non-target-site resistance (NTSR) to herbicides that disrupts agricultural weed control is a worldwide concern for food security. NTSR is considered a polygenic adaptive trait driven by differential gene regulation in resistant plants. Little is known about its genetic determinism, which precludes NTSR diagnosis and evolutionary studies. We used Illumina RNA-sequencing to investigate transcriptomic differences between plants from the global major weed rye-grass sensitive or resistant to the acetolactate-synthase (ALS) inhibiting herbicide pyroxsulam. Plants were collected before and along a time-course after herbicide application. De novo transcriptome assembly yielded a resource (LOLbase) including 92,381 contigs representing potentially active transcripts that were assigned putative annotations. Early effects of ALS inhibition consistent with the literature were observed in resistant and sensitive plants, proving LOLbase data were relevant to study herbicide response. Comparison of resistant and sensitive plants identified 30 candidate NTSR contigs. Further validation using 212 plants resistant or sensitive to pyroxsulam and/or to the ALS inhibitors iodosulfuron + mesosulfuron confirmed four contigs (two cytochromes P450, one glycosyl-transferase and one glutathione-S-transferase) were NTSR markers which combined expression levels could reliably identify resistant plants. This work confirmed that NTSR is driven by differential gene expression and involves different mechanisms. It provided tools and foundation for subsequent NTSR investigations.

  14. ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells

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    Jeron Andreas

    2012-12-01

    Full Text Available Abstract Background The transcription factor (TF forkhead box P3 (FOXP3 is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs. It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. Results ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin–stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Conclusions Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

  15. ChIP-on-chip analysis identifies IL-22 as direct target gene of ectopically expressed FOXP3 transcription factor in human T cells.

    Science.gov (United States)

    Jeron, Andreas; Hansen, Wiebke; Ewert, Franziska; Buer, Jan; Geffers, Robert; Bruder, Dunja

    2012-12-17

    The transcription factor (TF) forkhead box P3 (FOXP3) is constitutively expressed at high levels in naturally occurring CD4+CD25+ regulatory T cells (nTregs). It is not only the most accepted marker for that cell population but is also considered lineage determinative. Chromatin immunoprecipitation (ChIP) of TFs in combination with genomic tiling microarray analysis (ChIP-on-chip) has been shown to be an appropriate tool for identifying FOXP3 transcription factor binding sites (TFBSs) on a genome-wide scale. In combination with microarray expression analysis, the ChIP-on-chip technique allows identification of direct FOXP3 target genes. ChIP-on-chip analysis of the human FOXP3 expressed in resting and PMA/ionomycin-stimulated Jurkat T cells revealed several thousand putative FOXP3 binding sites and demonstrated the importance of intronic regions for FOXP3 binding. The analysis of expression data showed that the stimulation-dependent down-regulation of IL-22 was correlated with direct FOXP3 binding in the IL-22 promoter region. This association was confirmed by real-time PCR analysis of ChIP-DNA. The corresponding ChIP-region also contained a matching FOXP3 consensus sequence. Knowledge of the general distribution patterns of FOXP3 TFBSs in the human genome under resting and activated conditions will contribute to a better understanding of this TF and its influence on direct target genes, as well as its importance for the phenotype and function of Tregs. Moreover, FOXP3-dependent repression of Th17-related IL-22 may be relevant to an understanding of the phenomenon of Treg/Th17 cell plasticity.

  16. Kinome-wide RNAi studies in human multiple myeloma identify vulnerable kinase targets, including a lymphoid-restricted kinase, GRK6

    Science.gov (United States)

    Zhu, Yuan Xiao; Schmidt, Jessica; Yin, Hongwei; Shi, Chang-Xin; Que, Qiang; Basu, Gargi; Azorsa, David; Perkins, Louise M.; Braggio, Esteban; Fonseca, Rafael; Bergsagel, P. Leif; Mousses, Spyro; Stewart, A. Keith

    2010-01-01

    A paucity of validated kinase targets in human multiple myeloma has delayed clinical deployment of kinase inhibitors in treatment strategies. We therefore conducted a kinome-wide small interfering RNA (siRNA) lethality study in myeloma tumor lines bearing common t(4;14), t(14;16), and t(11;14) translocations to identify critically vulnerable kinases in myeloma tumor cells without regard to preconceived mechanistic notions. Fifteen kinases were repeatedly vulnerable in myeloma cells, including AKT1, AK3L1, AURKA, AURKB, CDC2L1, CDK5R2, FES, FLT4, GAK, GRK6, HK1, PKN1, PLK1, SMG1, and TNK2. Whereas several kinases (PLK1, HK1) were equally vulnerable in epithelial cells, others and particularly G protein–coupled receptor kinase, GRK6, appeared selectively vulnerable in myeloma. GRK6 inhibition was lethal to 6 of 7 myeloma tumor lines but was tolerated in 7 of 7 human cell lines. GRK6 exhibits lymphoid-restricted expression, and from coimmunoprecipitation studies we demonstrate that expression in myeloma cells is regulated via direct association with the heat shock protein 90 (HSP90) chaperone. GRK6 silencing causes suppression of signal transducer and activator of transcription 3 (STAT3) phosphorylation associated with reduction in MCL1 levels and phosphorylation, illustrating a potent mechanism for the cytotoxicity of GRK6 inhibition in multiple myeloma (MM) tumor cells. As mice that lack GRK6 are healthy, inhibition of GRK6 represents a uniquely targeted novel therapeutic strategy in human multiple myeloma. PMID:19996089

  17. A Patient-Derived, Pan-Cancer EMT Signature Identifies Global Molecular Alterations and Immune Target Enrichment Following Epithelial-to-Mesenchymal Transition.

    Science.gov (United States)

    Mak, Milena P; Tong, Pan; Diao, Lixia; Cardnell, Robert J; Gibbons, Don L; William, William N; Skoulidis, Ferdinandos; Parra, Edwin R; Rodriguez-Canales, Jaime; Wistuba, Ignacio I; Heymach, John V; Weinstein, John N; Coombes, Kevin R; Wang, Jing; Byers, Lauren Averett

    2016-02-01

    We previously demonstrated the association between epithelial-to-mesenchymal transition (EMT) and drug response in lung cancer using an EMT signature derived in cancer cell lines. Given the contribution of tumor microenvironments to EMT, we extended our investigation of EMT to patient tumors from 11 cancer types to develop a pan-cancer EMT signature. Using the pan-cancer EMT signature, we conducted an integrated, global analysis of genomic and proteomic profiles associated with EMT across 1,934 tumors including breast, lung, colon, ovarian, and bladder cancers. Differences in outcome and in vitro drug response corresponding to expression of the pan-cancer EMT signature were also investigated. Compared with the lung cancer EMT signature, the patient-derived, pan-cancer EMT signature encompasses a set of core EMT genes that correlate even more strongly with known EMT markers across diverse tumor types and identifies differences in drug sensitivity and global molecular alterations at the DNA, RNA, and protein levels. Among those changes associated with EMT, pathway analysis revealed a strong correlation between EMT and immune activation. Further supervised analysis demonstrated high expression of immune checkpoints and other druggable immune targets, such as PD1, PD-L1, CTLA4, OX40L, and PD-L2, in tumors with the most mesenchymal EMT scores. Elevated PD-L1 protein expression in mesenchymal tumors was confirmed by IHC in an independent lung cancer cohort. This new signature provides a novel, patient-based, histology-independent tool for the investigation of EMT and offers insights into potential novel therapeutic targets for mesenchymal tumors, independent of cancer type, including immune checkpoints. ©2015 American Association for Cancer Research.

  18. A patient-derived, pan-cancer EMT signature identifies global molecular alterations and immune target enrichment following epithelial to mesenchymal transition

    Science.gov (United States)

    Mak, Milena P.; Tong, Pan; Diao, Lixia; Cardnell, Robert J.; Gibbons, Don L.; William, William N.; Skoulidis, Ferdinandos; Parra, Edwin R.; Rodriguez-Canales, Jaime; Wistuba, Ignacio I.; Heymach, John V.; Weinstein, John N.; Coombes, Kevin R.; Wang, Jing; Byers, Lauren Averett

    2015-01-01

    Purpose We previously demonstrated the association between epithelial-to-mesenchymal transition (EMT) and drug response in lung cancer using an EMT signature derived in cancer cell lines. Given the contribution of tumor microenvironments to EMT, we extended our investigation of EMT to patient tumors from 11 cancer types to develop a pan-cancer EMT signature. Experimental Design Using the pan-cancer EMT signature, we conducted an integrated, global analysis of genomic and proteomic profiles associated with EMT across 1,934 tumors including breast, lung, colon, ovarian, and bladder cancers. Differences in outcome and in vitro drug response corresponding to expression of the pan-cancer EMT signature were also investigated. Results Compared to the lung cancer EMT signature, the patient-derived, pan-cancer EMT signature encompasses a set of core EMT genes that correlate even more strongly with known EMT markers across diverse tumor types and identifies differences in drug sensitivity and global molecular alterations at the DNA, RNA, and protein levels. Among those changes associated with EMT, pathway analysis revealed a strong correlation between EMT and immune activation. Further supervised analysis demonstrated high expression of immune checkpoints and other druggable immune targets such as PD1, PD-L1, CTLA4, OX40L, and PDL2, in tumors with the most mesenchymal EMT scores. Elevated PD-L1 protein expression in mesenchymal tumors was confirmed by immunohistochemistry in an independent lung cancer cohort. Conclusions This new signature provides a novel, patient-based, histology-independent tool for the investigation of EMT and offers insights into potential novel therapeutic targets for mesenchymal tumors, independent of cancer type, including immune checkpoints. PMID:26420858

  19. Zea mays(L.) P1 locus for cob glume color identified as a post-domestication selection target with an effect on temperate maize genomes

    Institute of Scientific and Technical Information of China (English)

    Chuanxiao; Xie; Jianfeng; Weng; Wenguo; Liu; Cheng; Zou; Zhuanfang; Hao; Wenxue; Li; Minshun; Li; Xiaosen; Guo; Gengyun; Zhang; Yunbi; Xu; Xinhai; Li; Shihuang; Zhang

    2013-01-01

    Artificial selection during domestication and post-domestication improvement results in loss of genetic diversity near target loci. However, the genetic locus associated with cob glume color and the nature of the genomic pattern surrounding it was elusive and the selection effect in that region was not clear. An association mapping panel consisting of 283 diverse modern temperate maize elite lines was genotyped by a chip containing over 55,000 evenly distributed SNPs. Ten-fold resequencing at the target region on 40 of the panel lines and 47 tropical lines was also undertaken. A genome-wide association study(GWAS) for cob glume color confirmed the P1 locus, which is located on the short arm of chromosome 1, with a-log10 P value for surrounding SNPs higher than the Bonferroni threshold(α/n, α < 0.001) when a mixed linear model(MLM) was implemented. A total of 26 markers were identified in a 0.78 Mb region surrounding the P1 locus, including 0.73 Mb and 0.05 Mb upstream and downstream of the P1 gene, respectively. A clear linkage disequilibrium(LD) block was found and LD decayed very rapidly with increasing physical distance surrounding the P1 locus. The estimates of π and Tajima’s D were significantly(P < 0.001) lower at both ends compared to the locus. Upon comparison of temperate and tropical lines at much finer resolution by resequencing(180-fold finer than chip SNPs), a more structured LD block pattern was found among the 40 resequenced temperate lines. All evidence indicates that the P1 locus in temperate maize has not undergone neutral evolution but has been subjected to artificial selection during post-domestication selection or improvement. The information and analytical results generated in this study provide insights as to how breeding efforts have affected genome evolution in crop plants.

  20. T cell post-transcriptional miRNA-mRNA interaction networks identify targets associated with susceptibility/resistance to collagen-induced arthritis.

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    Paula B Donate

    Full Text Available BACKGROUND: Due to recent studies indicating that the deregulation of microRNAs (miRNAs in T cells contributes to increased severity of rheumatoid arthritis, we hypothesized that deregulated miRNAs may interact with key mRNA targets controlling the function or differentiation of these cells in this disease. METHODOLOGY/PRINCIPAL FINDINGS: To test our hypothesis, we used microarrays to survey, for the first time, the expression of all known mouse miRNAs in parallel with genome-wide mRNAs in thymocytes and naïve and activated peripheral CD3(+ T cells from two mouse strains the DBA-1/J strain (MHC-H2q, which is susceptible to collagen induced arthritis (CIA, and the DBA-2/J strain (MHC-H2d, which is resistant. Hierarchical clustering of data showed the several T cell miRNAs and mRNAs differentially expressed between the mouse strains in different stages of immunization with collagen. Bayesian statistics using the GenMir(++ algorithm allowed reconstruction of post-transcriptional miRNA-mRNA interaction networks for target prediction. We revealed the participation of miR-500, miR-202-3p and miR-30b*, which established interactions with at least one of the following mRNAs: Rorc, Fas, Fasl, Il-10 and Foxo3. Among the interactions that were validated by calculating the minimal free-energy of base pairing between the miRNA and the 3'UTR of the mRNA target and luciferase assay, we highlight the interaction of miR-30b*-Rorc mRNA because the mRNA encodes a protein implicated in pro-inflammatory Th17 cell differentiation (Rorγt. FACS analysis revealed that Rorγt protein levels and Th17 cell counts were comparatively reduced in the DBA-2/J strain. CONCLUSIONS/SIGNIFICANCE: This result showed that the miRNAs and mRNAs identified in this study represent new candidates regulating T cell function and controlling susceptibility and resistance to CIA.

  1. Fine mapping of chromosome 5p15.33 based on a targeted deep sequencing and high density genotyping identifies novel lung cancer susceptibility loci.

    Science.gov (United States)

    Kachuri, Linda; Amos, Christopher I; McKay, James D; Johansson, Mattias; Vineis, Paolo; Bueno-de-Mesquita, H Bas; Boutron-Ruault, Marie-Christine; Johansson, Mikael; Quirós, J Ramón; Sieri, Sabina; Travis, Ruth C; Weiderpass, Elisabete; Le Marchand, Loic; Henderson, Brian E; Wilkens, Lynne; Goodman, Gary E; Chen, Chu; Doherty, Jennifer A; Christiani, David C; Wei, Yongyue; Su, Li; Tworoger, Shelley; Zhang, Xuehong; Kraft, Peter; Zaridze, David; Field, John K; Marcus, Michael W; Davies, Michael P A; Hyde, Russell; Caporaso, Neil E; Landi, Maria Teresa; Severi, Gianluca; Giles, Graham G; Liu, Geoffrey; McLaughlin, John R; Li, Yafang; Xiao, Xiangjun; Fehringer, Gord; Zong, Xuchen; Denroche, Robert E; Zuzarte, Philip C; McPherson, John D; Brennan, Paul; Hung, Rayjean J

    2016-01-01

    Chromosome 5p15.33 has been identified as a lung cancer susceptibility locus, however the underlying causal mechanisms were not fully elucidated. Previous fine-mapping studies of this locus have relied on imputation or investigated a small number of known, common variants. This study represents a significant advance over previous research by investigating a large number of novel, rare variants, as well as their underlying mechanisms through telomere length. Variants for this fine-mapping study were identified through a targeted deep sequencing (average depth of coverage greater than 4000×) of 576 individuals. Subsequently, 4652 SNPs, including 1108 novel SNPs, were genotyped in 5164 cases and 5716 controls of European ancestry. After adjusting for known risk loci, rs2736100 and rs401681, we identified a new, independent lung cancer susceptibility variant in LPCAT1: rs139852726 (OR = 0.46, P = 4.73×10(-9)), and three new adenocarcinoma risk variants in TERT: rs61748181 (OR = 0.53, P = 2.64×10(-6)), rs112290073 (OR = 1.85, P = 1.27×10(-5)), rs138895564 (OR = 2.16, P = 2.06×10(-5); among young cases, OR = 3.77, P = 8.41×10(-4)). In addition, we found that rs139852726 (P = 1.44×10(-3)) was associated with telomere length in a sample of 922 healthy individuals. The gene-based SKAT-O analysis implicated TERT as the most relevant gene in the 5p15.33 region for adenocarcinoma (P = 7.84×10(-7)) and lung cancer (P = 2.37×10(-5)) risk. In this largest fine-mapping study to investigate a large number of rare and novel variants within 5p15.33, we identified novel lung and adenocarcinoma susceptibility loci with large effects and provided support for the role of telomere length as the potential underlying mechanism.

  2. Fine mapping of chromosome 5p15.33 based on a targeted deep sequencing and high density genotyping identifies novel lung cancer susceptibility loci

    Science.gov (United States)

    Kachuri, Linda; Amos, Christopher I.; McKay, James D.; Johansson, Mattias; Vineis, Paolo; Bueno-de-Mesquita, H.Bas; Boutron-Ruault, Marie-Christine; Johansson, Mikael; Quirós, J.Ramón; Sieri, Sabina; Travis, Ruth C.; Weiderpass, Elisabete; Le Marchand, Loic; Henderson, Brian E.; Wilkens, Lynne; Goodman, Gary E.; Chen, Chu; Doherty, Jennifer A.; Christiani, David C.; Wei, Yongyue; Su, Li; Tworoger, Shelley; Zhang, Xuehong; Kraft, Peter; Zaridze, David; Field, John K.; Marcus, Michael W.; Davies, Michael P.A.; Hyde, Russell; Caporaso, Neil E.; Landi, Maria Teresa; Severi, Gianluca; Giles, Graham G.; Liu, Geoffrey; McLaughlin, John R.; Li, Yafang; Xiao, Xiangjun; Fehringer, Gord; Zong, Xuchen; Denroche, Robert E.; Zuzarte, Philip C.; McPherson, John D.; Brennan, Paul; Hung, Rayjean J.

    2016-01-01

    Chromosome 5p15.33 has been identified as a lung cancer susceptibility locus, however the underlying causal mechanisms were not fully elucidated. Previous fine-mapping studies of this locus have relied on imputation or investigated a small number of known, common variants. This study represents a significant advance over previous research by investigating a large number of novel, rare variants, as well as their underlying mechanisms through telomere length. Variants for this fine-mapping study were identified through a targeted deep sequencing (average depth of coverage greater than 4000×) of 576 individuals. Subsequently, 4652 SNPs, including 1108 novel SNPs, were genotyped in 5164 cases and 5716 controls of European ancestry. After adjusting for known risk loci, rs2736100 and rs401681, we identified a new, independent lung cancer susceptibility variant in LPCAT1: rs139852726 (OR = 0.46, P = 4.73×10–9), and three new adenocarcinoma risk variants in TERT: rs61748181 (OR = 0.53, P = 2.64×10–6), rs112290073 (OR = 1.85, P = 1.27×10–5), rs138895564 (OR = 2.16, P = 2.06×10–5; among young cases, OR = 3.77, P = 8.41×10–4). In addition, we found that rs139852726 (P = 1.44×10–3) was associated with telomere length in a sample of 922 healthy individuals. The gene-based SKAT-O analysis implicated TERT as the most relevant gene in the 5p15.33 region for adenocarcinoma (P = 7.84×10–7) and lung cancer (P = 2.37×10–5) risk. In this largest fine-mapping study to investigate a large number of rare and novel variants within 5p15.33, we identified novel lung and adenocarcinoma susceptibility loci with large effects and provided support for the role of telomere length as the potential underlying mechanism. PMID:26590902

  3. Targeted sequencing identifies associations between IL7R-JAK mutations and epigenetic modulators in T-cell acute lymphoblastic leukemia

    Science.gov (United States)

    Vicente, Carmen; Schwab, Claire; Broux, Michaël; Geerdens, Ellen; Degryse, Sandrine; Demeyer, Sofie; Lahortiga, Idoya; Elliott, Alannah; Chilton, Lucy; La Starza, Roberta; Mecucci, Cristina; Vandenberghe, Peter; Goulden, Nicholas; Vora, Ajay; Moorman, Anthony V.; Soulier, Jean; Harrison, Christine J.; Clappier, Emmanuelle; Cools, Jan

    2015-01-01

    T-cell acute lymphoblastic leukemia is caused by the accumulation of multiple oncogenic lesions, including chromosomal rearrangements and mutations. To determine the frequency and co-occurrence of mutations in T-cell acute lymphoblastic leukemia, we performed targeted re-sequencing of 115 genes across 155 diagnostic samples (44 adult and 111 childhood cases). NOTCH1 and CDKN2A/B were mutated/deleted in more than half of the cases, while an additional 37 genes were mutated/deleted in 4% to 20% of cases. We found that IL7R-JAK pathway genes were mutated in 27.7% of cases, with JAK3 mutations being the most frequent event in this group. Copy number variations were also detected, including deletions of CREBBP or CTCF and duplication of MYB. FLT3 mutations were rare, but a novel extracellular mutation in FLT3 was detected and confirmed to be transforming. Furthermore, we identified complex patterns of pairwise associations, including a significant association between mutations in IL7R-JAK genes and epigenetic regulators (WT1, PRC2, PHF6). Our analyses showed that IL7R-JAK genetic lesions did not confer adverse prognosis in T-cell acute lymphoblastic leukemia cases enrolled in the UK ALL2003 trial. Overall, these results identify interconnections between the T-cell acute lymphoblastic leukemia genome and disease biology, and suggest a potential clinical application for JAK inhibitors in a significant proportion of patients with T-cell acute lymphoblastic leukemia. PMID:26206799

  4. Targeted next generation sequencing of a panel of autism-related genes identifies an EHMT1 mutation in a Kleefstra syndrome patient with autism and normal intellectual performance.

    Science.gov (United States)

    Bock, István; Németh, Krisztina; Pentelényi, Klára; Balicza, Péter; Balázs, Anna; Molnár, Mária Judit; Román, Viktor; Nagy, József; Lévay, György; Kobolák, Julianna; Dinnyés, András

    2016-12-31

    Autism spectrum disorder (ASD) is a complex neurodevelopmental disorder with unknown genetic and environmental causation in most of the affected individuals. On the other hand, there are a growing number of ASD-associated syndromes, where the exact genetic origin can be revealed. Here we report a method, which included the targeted next generation sequencing (NGS) and filtering of 101 ASD associated genes, followed by database search. Next, RNA sequencing was used to study the region of interest at the transcriptional level. Using this workflow, we identified a de novo mutation in the euchromatic histone-lysine N-methyltransferase 1 gene (EHMT1) of an autistic patient with dysmorphisms. Sequencing of EHMT1 transcripts showed that the premature termination codon (Trp1138Ter) created by a single nucleotide change elicited nonsense-mediated mRNA decay, which led to haploinsufficiency already at the transcriptional level. Database and literature search provided evidence that this mutation caused Kleefstra syndrome (KS), which was confirmed by the presence of the disorder-specific phenotype in the patient. We provide a proof of principle that the implemented method is capable to elucidate the genetic etiology of individuals with syndromic autism. The novel mutation detected in the EHMT1 gene is responsible for KS's symptoms. In addition, further genetic factors might be involved in the ASD pathogenesis of the patient including a missense DPP6 mutation (Arg322Cys), which segregated with the autistic phenotype within the family.

  5. Novel HIV-1 knockdown targets identified by an enriched kinases/phosphatases shRNA library using a long-term iterative screen in Jurkat T-cells.

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    Sylvie Rato

    Full Text Available HIV-1 is a complex retrovirus that uses host machinery to promote its replication. Understanding cellular proteins involved in the multistep process of HIV-1 infection may result in the discovery of more adapted and effective therapeutic targets. Kinases and phosphatases are a druggable class of proteins critically involved in regulation of signal pathways of eukaryotic cells. Here, we focused on the discovery of kinases and phosphatases that are essential for HIV-1 replication but dispensable for cell viability. We performed an iterative screen in Jurkat T-cells with a short-hairpin-RNA (shRNA library highly enriched for human kinases and phosphatases. We identified 14 new proteins essential for HIV-1 replication that do not affect cell viability. These proteins are described to be involved in MAPK, JNK and ERK pathways, vesicular traffic and DNA repair. Moreover, we show that the proteins under study are important in an early step of HIV-1 infection before viral integration, whereas some of them affect viral transcription/translation. This study brings new insights for the complex interplay of HIV-1/host cell and opens new possibilities for antiviral strategies.

  6. Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

    Directory of Open Access Journals (Sweden)

    Britta Stadelmann

    2014-12-01

    Full Text Available A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ, a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

  7. Profound activity of the anti-cancer drug bortezomib against Echinococcus multilocularis metacestodes identifies the proteasome as a novel drug target for cestodes.

    Science.gov (United States)

    Stadelmann, Britta; Aeschbacher, Denise; Huber, Cristina; Spiliotis, Markus; Müller, Joachim; Hemphill, Andrew

    2014-12-01

    A library of 426 FDA-approved drugs was screened for in vitro activity against E. multilocularis metacestodes employing the phosphoglucose isomerase (PGI) assay. Initial screening at 20 µM revealed that 7 drugs induced considerable metacestode damage, and further dose-response studies revealed that bortezomib (BTZ), a proteasome inhibitor developed for the chemotherapy of myeloma, displayed high anti-metacestodal activity with an EC50 of 0.6 µM. BTZ treatment of E. multilocularis metacestodes led to an accumulation of ubiquinated proteins and unequivocally parasite death. In-gel zymography assays using E. multilocularis extracts demonstrated BTZ-mediated inhibition of protease activity in a band of approximately 23 kDa, the same size at which the proteasome subunit beta 5 of E. multilocularis could be detected by Western blot. Balb/c mice experimentally infected with E. multilocularis metacestodes were used to assess BTZ treatment, starting at 6 weeks post-infection by intraperitoneal injection of BTZ. This treatment led to reduced parasite weight, but to a degree that was not statistically significant, and it induced adverse effects such as diarrhea and neurological symptoms. In conclusion, the proteasome was identified as a drug target in E. multilocularis metacestodes that can be efficiently inhibited by BTZ in vitro. However, translation of these findings into in vivo efficacy requires further adjustments of treatment regimens using BTZ, or possibly other proteasome inhibitors.

  8. Use of Raman microscopy and band-target entropy minimization analysis to identify dyes in a commercial stamp. Implications for authentication and counterfeit detection.

    Science.gov (United States)

    Widjaja, Effendi; Garland, Marc

    2008-02-01

    Raman microscopy was used in mapping mode to collect more than 1000 spectra in a 100 microm x 100 microm area from a commercial stamp. Band-target entropy minimization (BTEM) was then employed to unmix the mixture spectra in order to extract the pure component spectra of the samples. Three pure component spectral patterns with good signal-to-noise ratios were recovered, and their spatial distributions were determined. The three pure component spectral patterns were then identified as copper phthalocyanine blue, calcite-like material, and yellow organic dye material by comparison to known spectral libraries. The present investigation, consisting of (1) advanced curve resolution (blind-source separation) followed by (2) spectral data base matching, readily suggests extensions to authenticity and counterfeit studies of other types of commercial objects. The presence or absence of specific observable components form the basis for assessment. The present spectral analysis (BTEM) is applicable to highly overlapping spectral information. Since a priori information such as the number of components present and spectral libraries are not needed in BTEM, and since minor signals arising from trace components can be reconstructed, this analysis offers a robust approach to a wide variety of material problems involving authenticity and counterfeit issues.

  9. Pan-Genome Analysis of Human Gastric Pathogen H. pylori: Comparative Genomics and Pathogenomics Approaches to Identify Regions Associated with Pathogenicity and Prediction of Potential Core Therapeutic Targets

    Directory of Open Access Journals (Sweden)

    Amjad Ali

    2015-01-01

    Full Text Available Helicobacter pylori is a human gastric pathogen implicated as the major cause of peptic ulcer and second leading cause of gastric cancer (~70% around the world. Conversely, an increased resistance to antibiotics and hindrances in the development of vaccines against H. pylori are observed. Pan-genome analyses of the global representative H. pylori isolates consisting of 39 complete genomes are presented in this paper. Phylogenetic analyses have revealed close relationships among geographically diverse strains of H. pylori. The conservation among these genomes was further analyzed by pan-genome approach; the predicted conserved gene families (1,193 constitute ~77% of the average H. pylori genome and 45% of the global gene repertoire of the species. Reverse vaccinology strategies have been adopted to identify and narrow down the potential core-immunogenic candidates. Total of 28 nonhost homolog proteins were characterized as universal therapeutic targets against H. pylori based on their functional annotation and protein-protein interaction. Finally, pathogenomics and genome plasticity analysis revealed 3 highly conserved and 2 highly variable putative pathogenicity islands in all of the H. pylori genomes been analyzed.

  10. A genome-wide RNAi screen in Caenorhabditis elegans identifies the nicotinic acetylcholine receptor subunit ACR-7 as an antipsychotic drug target.

    Directory of Open Access Journals (Sweden)

    Taixiang Saur

    Full Text Available We report a genome-wide RNA interference (RNAi screen for Suppressors of Clozapine-induced Larval Arrest (scla genes in Caenorhabditis elegans, the first genetic suppressor screen for antipsychotic drug (APD targets in an animal. The screen identifies 40 suppressors, including the α-like nicotinic acetylcholine receptor (nAChR homolog acr-7. We validate the requirement for acr-7 by showing that acr-7 knockout suppresses clozapine-induced larval arrest and that expression of a full-length translational GFP fusion construct rescues this phenotype. nAChR agonists phenocopy the developmental effects of clozapine, while nAChR antagonists partially block these effects. ACR-7 is strongly expressed in the pharynx, and clozapine inhibits pharyngeal pumping. acr-7 knockout and nAChR antagonists suppress clozapine-induced inhibition of pharyngeal pumping. These findings suggest that clozapine activates ACR-7 channels in pharyngeal muscle, leading to tetanus of pharyngeal muscle with consequent larval arrest. No APDs are known to activate nAChRs, but a number of studies indicate that α7-nAChR agonists may prove effective for the treatment of psychosis. α-like nAChR signaling is a mechanism through which clozapine may produce its therapeutic and/or toxic effects in humans, a hypothesis that could be tested following identification of the mammalian ortholog of C. elegans acr-7.

  11. Stathmin, a new target of PRL-3 identified by proteomic methods, plays a key role in progression and metastasis of colorectal cancer.

    Science.gov (United States)

    Zheng, Ping; Liu, Yong-Xia; Chen, Lin; Liu, Xun-Hua; Xiao, Zheng-Quan; Zhao, Liang; Li, Guang-Qiu; Zhou, Jun; Ding, Yan-Qing; Li, Jian-Ming

    2010-10-01

    To better understand the role of PRL-3 in progression and metastasis of colorectal cancer (CRC), we searched for PRL-3 associated proteins using proteomic methods. We identified 39 PRL-3 associated proteins based on proteomic strategy. Stathmin, a key oncoprotein, was proved to be a new PRL-3 associated protein. Notably, co-immunoprecipitation assays in both endogenous CRC cell lines and CRC tissues indicated that PRL-3 could interact with stathmin. And, both stathmin and PRL-3 contributed to microtubule (MT) destabilization of CRC cells. Moreover, gain-of-function and loss-of-function analyses revealed that stathmin promoted proliferation, cell adhesion, and migration of human CRC cells. Immunohistochemical analysis of 149 colorectal tumor samples showed that overexpression of stathmin was strongly correlated with tumor differentiation (P = 0.035), tumor invasion (P = 0.024), lymph node status (P PRL-3 associated proteins for the first time. The oncoprotein stathmin plays a key role in CRC as a new target of PRL-3. Interaction between PRL-3 and stathmin leads to MT destabilization of CRC cells, which contributes to progression and metastasis of CRC.

  12. MicroRNA fingerprints in juvenile myelomonocytic leukemia (JMML) identified miR-150-5p as a tumor suppressor and potential target for treatment.

    Science.gov (United States)

    Leoncini, Pier Paolo; Bertaina, Alice; Papaioannou, Dimitrios; Flotho, Christian; Masetti, Riccardo; Bresolin, Silvia; Menna, Giuseppe; Santoro, Nicola; Zecca, Marco; Basso, Giuseppe; Nigita, Giovanni; Veneziano, Dario; Pagotto, Sara; D'Ovidio, Katia; Rota, Rossella; Dorrance, Adrienne; Croce, Carlo M; Niemeyer, Charlotte; Locatelli, Franco; Garzon, Ramiro

    2016-08-23

    Juvenile myelomonocytic leukemia (JMML) is an aggressive leukemia of early childhood characterized by aberrant proliferation of myelomonocytic cells and hypersensitivity to GM-CSF stimulation. Mutually exclusive mutations in the RAS/ERK pathway genes such as PTPN11, NRAS, KRAS, CBL, or NF1 are found in ~90% of the cases. These mutations give rise to disease at least in part by activating STAT5 through phosphorylation and by promoting cell growth. MicroRNAs (miRs) are small non-coding RNAs that regulate gene expression, which are often deregulated in leukemia. However, little is known about their role in JMML. Here, we report distinctive miR expression signatures associated with the molecular subgroups of JMML. Among the downregulated miRs in JMML, miR-150-5p was found to target STAT5b, a gene which is often over-activated in JMML, and contributes to the characteristic aberrant signaling of this disorder. Moreover, loss of miR-150-5p and upregulation of STAT5b expression were also identified in a murine model of JMML. Ectopic overexpression of miR-150-5p in mononuclear cells from three JMML patients significantly decreased cell proliferation. Altogether, our data indicate that miR expression is deregulated in JMML and may play a role in the pathogenesis of this disorder by modulating key effectors of cytokine receptor pathways.

  13. Expression analyses identify MLL as a prominent target of 11q23 amplification and support an etiologic role for MLL gain of function in myeloid malignancies.

    Science.gov (United States)

    Poppe, Bruce; Vandesompele, Jo; Schoch, Claudia; Lindvall, Charlotta; Mrozek, Krzysztof; Bloomfield, Clara D; Beverloo, H Berna; Michaux, Lucienne; Dastugue, Nicole; Herens, Christian; Yigit, Nurten; De Paepe, Anne; Hagemeijer, Anne; Speleman, Frank

    2004-01-01

    MLL amplification was recently recognized as a recurrent aberration in acute myeloid leukemia (AML) and myelodys-plastic syndrome (MDS), associated with adverse prognosis and karyotype complexity. Here we present detailed results of fluorescence in situ hybridization (FISH) and expression analyses of MLL and 5 selected 11q candidate oncogenes (CBL, DDX6, ETS1, FLI1, and PLZF) in 31 patient samples and one cell line with 11q23 gain. FISH analyses revealed that the 11q23 amplicon invariably encompassed MLL, DDX6, ETS1, and FLI1, whereas expression analyses identified MLL and DDX6 as the most differentially expressed genes among samples with and without 11q23 copy gain or amplification. In MLL-amplified samples, a significant transcriptional up-regulation of MEIS1, PROML1, ADAM10, NKG2D, and ITPA was noted. Further analyses, designed to elucidate a possible role of the 11q overexpressed genes (MLL, DDX6, FLI1, and ETS1) in unselected MDS and AML samples, revealed a significant upregulation of MLL in MDS. Our findings confirm the MLL gene as a prominent target of 11q23 amplification and provide further evidence for an etiologic role for MLL gain of function in myeloid malignancies. In addition, our results indicate that the transcriptional program associated with MLL rearrangements and MLL overexpression displays significant similarities.

  14. Identifying cost-minimizing strategies for guaranteeing target dairy income over feed cost via use of the Livestock Gross Margin dairy insurance program.

    Science.gov (United States)

    Valvekar, M; Cabrera, V E; Gould, B W

    2010-07-01

    under the cost-minimizing solution representing 1.9% of the insured amount. Our model identifies the lowest cost insurance contract for a desired target guaranteed income over feed cost. Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

  15. A genomic approach to therapeutic target validation identifies a glucose-lowering GLP1R variant protective for coronary heart disease

    DEFF Research Database (Denmark)

    Scott, Robert A; Freitag, Daniel F; Li, Li

    2016-01-01

    therefore investigated the association of variants in six genes that encode drug targets for obesity or T2D with a range of metabolic traits in up to 11,806 individuals by targeted exome sequencing and follow-up in 39,979 individuals by targeted genotyping, with additional in silico follow-up in consortia...... in randomized controlled trials. Genetic variants associated with metabolic traits and multiple disease outcomes can be used to validate therapeutic targets at an early stage in the drug development process....

  16. A novel highly divergent protein family identified from a viviparous insect by RNA-seq analysis: a potential target for tsetse fly-specific abortifacients.

    Directory of Open Access Journals (Sweden)

    Joshua B Benoit

    2014-04-01

    Full Text Available In tsetse flies, nutrients for intrauterine larval development are synthesized by the modified accessory gland (milk gland and provided in mother's milk during lactation. Interference with at least two milk proteins has been shown to extend larval development and reduce fecundity. The goal of this study was to perform a comprehensive characterization of tsetse milk proteins using lactation-specific transcriptome/milk proteome analyses and to define functional role(s for the milk proteins during lactation. Differential analysis of RNA-seq data from lactating and dry (non-lactating females revealed enrichment of transcripts coding for protein synthesis machinery, lipid metabolism and secretory proteins during lactation. Among the genes induced during lactation were those encoding the previously identified milk proteins (milk gland proteins 1-3, transferrin and acid sphingomyelinase 1 and seven new genes (mgp4-10. The genes encoding mgp2-10 are organized on a 40 kb syntenic block in the tsetse genome, have similar exon-intron arrangements, and share regions of amino acid sequence similarity. Expression of mgp2-10 is female-specific and high during milk secretion. While knockdown of a single mgp failed to reduce fecundity, simultaneous knockdown of multiple variants reduced milk protein levels and lowered fecundity. The genomic localization, gene structure similarities, and functional redundancy of MGP2-10 suggest that they constitute a novel highly divergent protein family. Our data indicates that MGP2-10 function both as the primary amino acid resource for the developing larva and in the maintenance of milk homeostasis, similar to the function of the mammalian casein family of milk proteins. This study underscores the dynamic nature of the lactation cycle and identifies a novel family of lactation-specific proteins, unique to Glossina sp., that are essential to larval development. The specificity of MGP2-10 to tsetse and their critical role during

  17. Targeted Proteomics Identifies Paraoxonase/Arylesterase 1 (PON1) and Apolipoprotein Cs as Potential Risk Factors for Hypoalphalipoproteinemia in Diabetic Subjects Treated with Fenofibrate and Rosiglitazone*

    Science.gov (United States)

    Ronsein, Graziella E.; Reyes-Soffer, Gissette; He, Yi; Oda, Michael; Ginsberg, Henry; Heinecke, Jay W.

    2016-01-01

    Low levels of high-density lipoprotein cholesterol (HDL-C) and high triglyceride levels contribute to the excess rate of cardiovascular events seen in subjects with type 2 diabetes. Fenofibrate treatment partially reverses dyslipidemia in these subjects. However, a paradoxical marked reduction in HDL-C and HDL's major protein, apolipoprotein A-I, is a complication of fenofibrate in combination with rosiglitazone, an insulin-sensitizing agent. Risk factors for this condition, termed hypoalphalipoproteinemia, have yet to be identified. Using a case-control study design with subjects enrolled in the Action to Control Cardiovascular Risk in Diabetes (ACCORD) trial, we tested the hypothesis that alterations in HDL's protein cargo predispose diabetic subjects to fenofibrate/rosiglitazone-induced hypoalphalipoproteinemia. HDL was isolated from blood obtained from controls (no decreases or increase in HDL-C while receiving fenofibrate/rosiglitazone therapy) and cases (developed hypoalphalipoproteinemia after fenofibrate/rosiglitazone treatment) participating in the ACCORD study before they began fenofibrate/rosiglitazone treatment. HDL proteins were quantified by targeted parallel reaction monitoring (PRM) and selected reaction monitoring (SRM) with isotope dilution. This approach demonstrated marked increases in the relative concentrations of paraoxonase/arylesterase 1 (PON1), apolipoprotein C-II (APOC2), apolipoprotein C-I, and apolipoprotein H in the HDL of subjects who developed hypoalphalipoproteinemia. The case and control subjects did not differ significantly in baseline HDL-C levels or other traditional lipid risk factors. We used orthogonal biochemical techniques to confirm increased levels of PON1 and APOC2. Our observations suggest that an imbalance in HDL proteins predisposes diabetic subjects to develop hypoalphalipoproteinemia on fenofibrate/rosiglitazone therapy. PMID:26667175

  18. Peptide microarray profiling identifies phospholipase C gamma 1 (PLC-γ1) as a potential target for t(8;21) AML

    Science.gov (United States)

    Mahmud, Hasan; Scherpen, Frank J.G.; de Boer, Tiny Meeuwsen; Lourens, Harm-Jan; Schoenherr, Caroline; Eder, Matthias; Scherr, Michaela; Guryev, Victor; De Bont, Eveline S.

    2017-01-01

    The t(8;21) (q22;q22) chromosomal translocation is one of the most frequent genetic alterations in acute myeloid leukemia (AML) which has a need for improved therapeutic strategies. We found PLC-γ1 as one of the highest phosphorylated peptides in t(8;21) AML samples compared to NBM or CN-AML in our previous peptide microarray. PLC-γ1 is known to play a role in cancer progression, however, the impact of PLC-γ1 in AML is currently unknown. Therefore, we aimed to study the functional role of PLC-γ1 by investigating the cellular growth, survival and its underlying mechanism in t(8;21) AML. In this study, PLC-γ1 expression was significantly higher in t(8;21) AML compared to other karyotypes. The PLC-γ1 protein expression was suppressed in AML1-ETO knock down cells indicating that it might induce kasumi-1 cell death. ShRNA-mediated PLC-γ1 knockdown in kasumi-1 cells significantly blocked cell growth, induced apoptosis and cell cycle arrest which was explained by the increased activation of apoptotic related and cell cycle regulatory protein expressions. Gene expression array analysis showed the up-regulation of apoptotic and DNA damage response genes together with the downregulation of cell growth, proliferation and differentiation genes in the PLC-γ1 suppressed kasumi-1 cells, consistent with the observed phenotypic effects. Importantly, PLC-γ1 suppressed kasumi-1 cells showed higher chemosensitivity to the chemotherapeutic drug treatments and lower cell proliferation upon hypoxic stress. Taken together, these in vitro finding strongly support an important role for PLC-γ1 in the survival of t(8;21) AML mimicking kasumi-1 cells and identify PLC-γ1 as a potential therapeutic target for t(8;21) AML treatment.

  19. Impaired plasma membrane targeting or protein stability by certain ATP1A2 mutations identified in sporadic or familial hemiplegic migraine.

    Science.gov (United States)

    Tavraz, Neslihan N; Dürr, Katharina L; Koenderink, Jan B; Freilinger, Tobias; Bamberg, Ernst; Dichgans, Martin; Friedrich, Thomas

    2009-01-01

    Mutations in three different genes have been implicated in familial hemiplegic migraine (FHM), two of them code for neuronal voltage-gated cation channels, CACNA1A and SCN1A, while the third encodes ATP1A2, the alpha(2)-isoform of the Na(+)/K(+)-ATPase's catalytic subunit, thus classifying FHM as an ion channel/ion transporter disorder. The Na(+)/K(+)-ATPase maintains the physiological gradients for Na(+) and K(+) ions and is therefore critical for the activity of ion channels and transporters involved in neurotransmitter uptake or Ca(2+) signaling. Diverse functional abnormalities have been identified for disease-linked ATP1A2 mutations, which reach far beyond simple loss-of-function. We have shown recently that ATP1A2 mutations frequently lead to changes in the enzyme's voltage-dependent properties, kinetics or apparent cation affinities. Here, we present functional data on a so far uncharacterized set of ATP1A2 mutations (G301R, R908Q and P979L) upon expression in Xenopus oocytes and HEK293FT cells, and provide evidence for a novel pathophysiological mechanism. Whereas the G301R mutant was inactive, no functional changes were observed for mutants R908Q and P979L in the oocyte expression system. However, the R908Q mutant was less effectively expressed in the plasma membrane of oocytes, making it the first missense mutation to result in defective plasma membrane targeting. Notably, the P979L mutant exhibited the same cellular expression profile as the wild-type protein, both in Xenopus oocytes and in transfected HEK293FT cells grown at 28 degrees C, but much less P979L protein was found upon cell growth at 37 degrees C, showing for the first time that temperature-sensitive effects on protein stability can underlie ATP1A2 loss-of-function.

  20. Mass spectrometric characterization of urinary metabolites of the selective androgen receptor modulator S-22 to identify potential targets for routine doping controls.

    Science.gov (United States)

    Thevis, Mario; Thomas, Andreas; Möller, Ines; Geyer, Hans; Dalton, James T; Schänzer, Wilhelm

    2011-08-15

    Drugs that promote anabolic processes with limited undesirable effects are of considerable therapeutic interest; some notable examples include those for the treatment of cancer cachexia and muscle-wasting diseases. Anabolic properties are not only therapeutically beneficial to critically ill and debilitated patients, but are also desirable to athletes seeking artificial enhancements in endurance, strength and accelerated recovery. The use of anabolic agents in the clinical setting is being reconsidered with the emergence of a new class of drugs referred to as SARMs (selective androgen receptor modulators). SARMs have the potential to complement or even replace anabolic androgenic steroidal use with the benefit of a reduction of the undesirable side effects associated with steroid administration alone. Arylpropionamide-based SARMs such as andarine (S-4) and S-22 have shown promising therapeutic properties and have attracted the interest of elite and amateur athletes despite the absence of clinical approval, and evidence for trafficking and misuse in sport has been obtained by doping control authorities. In this communication, the elucidation of urinary metabolites of the SARM drug candidate S-22 is compared with earlier in vitro metabolism studies. Following oral administration of illicit S-22, urine samples were collected after 62 and 135 h and analyzed for the active drug and its major metabolic products. Liquid chromatography interfaced with high-resolution/high-accuracy (tandem) mass spectrometry was used to identify and/or confirm the predicted target analytes for sports drug testing purposes. S-22 was detected in both specimens accompanied by its glucuronic acid conjugate. This was the B-ring hydroxylated derivative of S-22 plus the corresponding glucuronide (with the phase-II metabolites being the more abundant analytes). In addition, the samples collected 62 h post-administration also contained the phase-I metabolite hydroxylated at the methyl residue (C-20

  1. DNA methylation profiling of ovarian carcinomas and their in vitro models identifies HOXA9, HOXB5, SCGB3A1, and CRABP1 as novel targets

    Directory of Open Access Journals (Sweden)

    Tropé Claes G

    2007-07-01

    Full Text Available Abstract Background The epigenetics of ovarian carcinogenesis remains poorly described. We have in the present study investigated the promoter methylation status of 13 genes in primary ovarian carcinomas (n = 52 and their in vitro models (n = 4; ES-2, OV-90, OVCAR-3, and SKOV-3 by methylation-specific polymerase chain reaction (MSP. Direct bisulphite sequencing analysis was used to confirm the methylation status of individual genes. The MSP results were compared with clinico- pathological features. Results Eight out of the 13 genes were hypermethylated among the ovarian carcinomas, and altogether 40 of 52 tumours were methylated in one or more genes. Promoter hypermethylation of HOXA9, RASSF1A, APC, CDH13, HOXB5, SCGB3A1 (HIN-1, CRABP1, and MLH1 was found in 51% (26/51, 49% (23/47, 24% (12/51, 20% (10/51, 12% (6/52, 10% (5/52, 4% (2/48, and 2% (1/51 of the carcinomas, respectively, whereas ADAMTS1, MGMT, NR3C1, p14ARF, and p16INK4a were unmethylated in all samples. The methylation frequencies of HOXA9 and SCGB3A1 were higher among relatively early-stage carcinomas (FIGO I-II than among carcinomas of later stages (FIGO III-IV; P = 0.002, P = 0.020, respectively. The majority of the early-stage carcinomas were of the endometrioid histotype. Additionally, HOXA9 hypermethylation was more common in tumours from patients older than 60 years of age (15/21 than among those of younger age (11/30; P = 0.023. Finally, there was a significant difference in HOXA9 methylation frequency among the histological types (P = 0.007. Conclusion DNA hypermethylation of tumour suppressor genes seems to play an important role in ovarian carcinogenesis and HOXA9, HOXB5, SCGB3A1, and CRABP1 are identified as novel hypermethylated target genes in this tumour type.

  2. Computational prediction of the Crc regulon identifies genus-wide and species-specific targets of catabolite repression control in Pseudomonas bacteria

    LENUS (Irish Health Repository)

    Browne, Patrick

    2010-11-25

    Abstract Background Catabolite repression control (CRC) is an important global control system in Pseudomonas that fine tunes metabolism in order optimise growth and metabolism in a range of different environments. The mechanism of CRC in Pseudomonas spp. centres on the binding of a protein, Crc, to an A-rich motif on the 5\\' end of an mRNA resulting in translational down-regulation of target genes. Despite the identification of several Crc targets in Pseudomonas spp. the Crc regulon has remained largely unexplored. Results In order to predict direct targets of Crc, we used a bioinformatics approach based on detection of A-rich motifs near the initiation of translation of all protein-encoding genes in twelve fully sequenced Pseudomonas genomes. As expected, our data predict that genes related to the utilisation of less preferred nutrients, such as some carbohydrates, nitrogen sources and aromatic carbon compounds are targets of Crc. A general trend in this analysis is that the regulation of transporters is conserved across species whereas regulation of specific enzymatic steps or transcriptional activators are often conserved only within a species. Interestingly, some nucleoid associated proteins (NAPs) such as HU and IHF are predicted to be regulated by Crc. This finding indicates a possible role of Crc in indirect control over a subset of genes that depend on the DNA bending properties of NAPs for expression or repression. Finally, some virulence traits such as alginate and rhamnolipid production also appear to be regulated by Crc, which links nutritional status cues with the regulation of virulence traits. Conclusions Catabolite repression control regulates a broad spectrum of genes in Pseudomonas. Some targets are genus-wide and are typically related to central metabolism, whereas other targets are species-specific, or even unique to particular strains. Further study of these novel targets will enhance our understanding of how Pseudomonas bacteria integrate

  3. Using consensus-shape clustering to identify promiscuous ligands and protein targets and to choose the right query for shape-based virtual screening.

    Science.gov (United States)

    Pérez-Nueno, Violeta I; Ritchie, David W

    2011-06-27

    Ligand-based shape matching approaches have become established as important and popular virtual screening (VS) techniques. However, despite their relative success, many authors have discussed how best to choose the initial query compounds and which of their conformations should be used. Furthermore, it is increasingly the case that pharmaceutical companies have multiple ligands for a given target and these may bind in different ways to the same pocket. Conversely, a given ligand can sometimes bind to multiple targets, and this is clearly of great importance when considering drug side-effects. We recently introduced the notion of spherical harmonic-based "consensus shapes" to help deal with these questions. Here, we apply a consensus shape clustering approach to the 40 protein-ligand targets in the DUD data set using PARASURF/PARAFIT. Results from clustering show that in some cases the ligands for a given target are split into two subgroups which could suggest they bind to different subsites of the same target. In other cases, our clustering approach sometimes groups together ligands from different targets, and this suggests that those ligands could bind to the same targets. Hence spherical harmonic-based clustering can rapidly give cross-docking information while avoiding the expense of performing all-against-all docking calculations. We also report on the effect of the query conformation on the performance of shape-based screening of the DUD data set and the potential gain in screening performance by using consensus shapes calculated in different ways. We provide details of our analysis of shape-based screening using both PARASURF/PARAFIT and ROCS, and we compare the results obtained with shape-based and conventional docking approaches using MSSH/SHEF and GOLD. The utility of each type of query is analyzed using commonly reported statistics such as enrichment factors (EF) and receiver-operator-characteristic (ROC) plots as well as other early performance metrics.

  4. Yeast Augmented Network Analysis (YANA: a new systems approach to identify therapeutic targets for human genetic diseases [v1; ref status: indexed, http://f1000r.es/3gk

    Directory of Open Access Journals (Sweden)

    David J. Wiley

    2014-06-01

    Full Text Available Genetic interaction networks that underlie most human diseases are highly complex and poorly defined. Better-defined networks will allow identification of a greater number of therapeutic targets. Here we introduce our Yeast Augmented Network Analysis (YANA approach and test it with the X-linked spinal muscular atrophy (SMA disease gene UBA1. First, we express UBA1 and a mutant variant in fission yeast and use high-throughput methods to identify fission yeast genetic modifiers of UBA1. Second, we analyze available protein-protein interaction network databases in both fission yeast and human to construct UBA1 genetic networks. Third, from these networks we identified potential therapeutic targets for SMA. Finally, we validate one of these targets in a vertebrate (zebrafish SMA model. This study demonstrates the power of combining synthetic and chemical genetics with a simple model system to identify human disease gene networks that can be exploited for treating human diseases.

  5. INTRAUTERINE ADHESIONS IN ABUJA, NIGERIA.

    African Journals Online (AJOL)

    Context: Infertility and menstrual abnormalities continue to constitute a significant bulk of gynaecological consultations. Both of these ... IUA can be diagnosed through various methods. ... exogenous steroid hormone therapy to regenerate the.

  6. Phosphoproteomics identified Endofin, DCBLD2, and KIAA0582 as novel tyrosine phosphorylation targets of EGF signaling and Iressa in human cancer cells

    DEFF Research Database (Denmark)

    Chen, Yunhao; Low, Teck-Yew; Choong, Lee-Yee;

    2007-01-01

    and relatively quantified the tyrosine phosphorylation levels of 21 proteins between control and EGF-treated A431 human cervical cancer cells. Of these, Endofin, DCBLD2, and KIAA0582 were validated to be novel tyrosine-phosphorylation targets of EGF signaling and Iressa, a highly selective inhibitor of EGFR...

  7. Functional Proteomics Identifies Acinus L as a Direct Insulin- and Amino Acid-Dependent Mammalian Target of Rapamycin Complex 1 (mTORC1) Substrate

    NARCIS (Netherlands)

    Schwarz, Jennifer Jasmin; Wiese, Heike; Toelle, Regine Charlotte; Zarei, Mostafa; Dengjel, Joern; Warscheid, Bettina; Thedieck, Kathrin

    2015-01-01

    The serine/threonine kinase mammalian target of rapamycin (mTOR) governs growth, metabolism, and aging in response to insulin and amino acids (aa), and is often activated in metabolic disorders and cancer. Much is known about the regulatory signaling network that encompasses mTOR, but surprisingly f

  8. Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization

    NARCIS (Netherlands)

    Zych, Courtney; Dömling, A.; Ayyavoo, Velpandi

    2013-01-01

    Targeting protein-protein interactions (PPI) is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV)-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of H

  9. Identifying Consumption Target in Microblog Based on Cross Social Media Search Identifying consumption target in microblog based on cross social media search%基于跨社交媒体检索的微博消费对象识别

    Institute of Scientific and Technical Information of China (English)

    付博; 刘挺

    2015-01-01

    目前,微博消费意图识别问题成为新的研究热点。然而,已有工作主要判断微博是否具有商业意图,很少研究消费意图内容中消费对象的识别问题,而消费对象的识别是精确地进行商业推荐的关键,因此对其进行研究具有重要意义。微博内容较短且用户用语不规范,基于词典匹配与依存关系分析的方法,在识别召回率上难以得到满意的效果。提出了一种基于跨媒体伪相关反馈的方法。首先分析具有消费意图微博中的关键词,而后将其视为查询提交给搜索引擎进行搜索,最后从搜索结果中提取消费意图对象。实验结果表明,提出的方法显著地优于基准方法。%Consumption intention recognition in microblog is a new research topic in social media. However, existing work mainly focuses on identifying the commercial intention in microblog, literatures engaged in the recognition of consumption target are still sparse. Meanwhile, recognizing consumption target is the key to accurate commercial recommendation. Since the contents in microblog are usually short and informally using of terms, the results of dic-tionary matching and dependency analysis based methods are not satisfied as the low recall. This paper presents a new method which is based on pseudo relevance feedback by using cross media. This paper firstly analyzes the key-words in consumption intention, then formulates it as queries to submit to the search engine, finally extracts consumer target from the search results. The experimental results are significantly better than those of the baseline methods relying on existing technologies.

  10. An Algorithm to Identify Target-Selective Ligands – A Case Study of 5-HT7/5-HT1A Receptor Selectivity

    Science.gov (United States)

    Kurczab, Rafał; Canale, Vittorio; Zajdel, Paweł; Bojarski, Andrzej J.

    2016-01-01

    A computational procedure to search for selective ligands for structurally related protein targets was developed and verified for serotonergic 5-HT7/5-HT1A receptor ligands. Starting from a set of compounds with annotated activity at both targets (grouped into four classes according to their activity: selective toward each target, not-selective and not-selective but active) and with an additional set of decoys (prepared using DUD methodology), the SVM (Support Vector Machines) models were constructed using a selective subset as positive examples and four remaining classes as negative training examples. Based on these four component models, the consensus classifier was then constructed using a data fusion approach. The combination of two approaches of data representation (molecular fingerprints vs. structural interaction fingerprints), different training set sizes and selection of the best SVM component models for consensus model generation, were evaluated to determine the optimal settings for the developed algorithm. The results showed that consensus models with molecular fingerprints, a larger training set and the selection of component models based on MCC maximization provided the best predictive performance. PMID:27271158

  11. An Algorithm to Identify Target-Selective Ligands - A Case Study of 5-HT7/5-HT1A Receptor Selectivity.

    Directory of Open Access Journals (Sweden)

    Rafał Kurczab

    Full Text Available A computational procedure to search for selective ligands for structurally related protein targets was developed and verified for serotonergic 5-HT7/5-HT1A receptor ligands. Starting from a set of compounds with annotated activity at both targets (grouped into four classes according to their activity: selective toward each target, not-selective and not-selective but active and with an additional set of decoys (prepared using DUD methodology, the SVM (Support Vector Machines models were constructed using a selective subset as positive examples and four remaining classes as negative training examples. Based on these four component models, the consensus classifier was then constructed using a data fusion approach. The combination of two approaches of data representation (molecular fingerprints vs. structural interaction fingerprints, different training set sizes and selection of the best SVM component models for consensus model generation, were evaluated to determine the optimal settings for the developed algorithm. The results showed that consensus models with molecular fingerprints, a larger training set and the selection of component models based on MCC maximization provided the best predictive performance.

  12. Integrative ChIP-seq/microarray analysis identifies a CTNNB1 target signature enriched in intestinal stem cells and colon cancer.

    Directory of Open Access Journals (Sweden)

    Kazuhide Watanabe

    Full Text Available BACKGROUND: Deregulation of canonical Wnt/CTNNB1 (beta-catenin pathway is one of the earliest events in the pathogenesis of colon cancer. Mutations in APC or CTNNB1 are highly frequent in colon cancer and cause aberrant stabilization of CTNNB1, which activates the transcription of Wnt target genes by binding to chromatin via the TCF/LEF transcription factors. Here we report an integrative analysis of genome-wide chromatin occupancy of CTNNB1 by chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq and gene expression profiling by microarray analysis upon RNAi-mediated knockdown of CTNNB1 in colon cancer cells. RESULTS: We observed 3629 CTNNB1 binding peaks across the genome and a significant correlation between CTNNB1 binding and knockdown-induced gene expression change. Our integrative analysis led to the discovery of a direct Wnt target signature composed of 162 genes. Gene ontology analysis of this signature revealed a significant enrichment of Wnt pathway genes, suggesting multiple feedback regulations of the pathway. We provide evidence that this gene signature partially overlaps with the Lgr5+ intestinal stem cell signature, and is significantly enriched in normal intestinal stem cells as well as in clinical colorectal cancer samples. Interestingly, while the expression of the CTNNB1 target gene set does not correlate with survival, elevated expression of negative feedback regulators within the signature predicts better prognosis. CONCLUSION: Our data provide a genome-wide view of chromatin occupancy and gene regulation of Wnt/CTNNB1 signaling in colon cancer cells.

  13. MHC class I-presented T cell epitopes identified by immunoproteomics analysis are targets for a cross reactive influenza-specific T cell response.

    Directory of Open Access Journals (Sweden)

    James S Testa

    Full Text Available Influenza virus infection and the resulting complications are a significant global public health problem. Improving humoral immunity to influenza is the target of current conventional influenza vaccines, however, these are generally not cross-protective. On the contrary, cell-mediated immunity generated by primary influenza infection provides substantial protection against serologically distinct viruses due to recognition of cross-reactive T cell epitopes, often from internal viral proteins conserved between viral subtypes. Efforts are underway to develop a universal flu vaccine that would stimulate both the humoral and cellular immune responses leading to long-lived memory. Such a universal vaccine should target conserved influenza virus antibody and T cell epitopes that do not vary from strain to strain. In the last decade, immunoproteomics, or the direct identification of HLA class I presented epitopes, has emerged as an alternative to the motif prediction method for the identification of T cell epitopes. In this study, we used this method to uncover several cross-specific MHC class I specific T cell epitopes naturally presented by influenza A-infected cells. These conserved T cell epitopes, when combined with a cross-reactive antibody epitope from the ectodomain of influenza M2, generate cross-strain specific cell mediated and humoral immunity. Overall, we have demonstrated that conserved epitope-specific CTLs could recognize multiple influenza strain infected target cells and, when combined with a universal antibody epitope, could generate virus specific humoral and T cell responses, a step toward a universal vaccine concept. These epitopes also have potential as new tools to characterize T cell immunity in influenza infection, and may serve as part of a universal vaccine candidate complementary to current vaccines.

  14. Expression of Erk5 in early stage breast cancer and association with disease free survival identifies this kinase as a potential therapeutic target.

    Directory of Open Access Journals (Sweden)

    Juan Carlos Montero

    Full Text Available BACKGROUND: Breast cancer is the most common neoplasia in women. Even though advances in its treatment have improved disease outcome, some patients relapse. Therefore, attempts to better define the molecular determinants that drive breast cancer cell proliferation may help in defining potential therapeutic targets. Mitogen-activated protein kinases (MAPK play important roles in tumorigenesis. One of them, Erk5, has been linked to the proliferation of breast cancer cells in vitro. Here we have investigated the expression and prognostic value of Erk5 in human breast cancer. METHODOLOGY/PRINCIPAL FINDINGS: Animal and cellular models were used to study Erk5 expression and function in breast cancer. In 84 human breast tumours the expression of Erk5 was analyzed by immunohistochemistry. Active Erk5 (pErk5 was studied by Western blotting. Correlation of Erk5 with clinicopathological parameters and with disease-free survival in early stage breast cancer patients was analyzed. Expression of Erk5 was detected in most patients, and overexpression was found in 20%. Active Erk5 was present in a substantial number of samples, as well as in tumours from an animal breast cancer model. Overexpression of Erk5 was associated with a decrease in disease-free survival time, which was independent of other clinicopathological parameters of prognosis. Transient transfection of a short hairpin RNA (shRNA targeting Erk5, and a stable cell line expressing a dominant negative form of Erk5 (Erk5(AEF, were used to investigate the influence of Erk5 on drugs used in the clinic to treat breast tumours. We found that inhibition of Erk5 decreased cancer cell proliferation and also sensitized these cells to the action of anti-HER2 therapies. CONCLUSIONS/SIGNIFICANCE: Overexpression of Erk5 is an independent predictor of disease-free survival in breast cancer, and may represent a future therapeutic target.

  15. TARGET Researchers Identify Mutations in SIX1/2 and microRNA Processing Genes in Favorable Histology Wilms Tumor | Office of Cancer Genomics

    Science.gov (United States)

    TARGET researchers molecularly characterized favorable histology Wilms tumor (FHWT), a pediatric renal cancer. Comprehensive genome and transcript analyses revealed single-nucleotide substitution/deletion mutations in microRNA processing genes (15% of FHWT patients) and Sine Oculis Homeobox Homolog 1/2 (SIX1/2) genes (7% of FHWT patients). SIX1/2 genes play a critical role in renal development and were not previously associated with FHWT, thus presenting a novel role for SIX1/2 pathway aberrations in this disease.

  16. SILAC-based quantitative proteomics identified lysosome as a fast response target to PDT agent Gd-N induced oxidative stress in human ovarian cancer IGROV1 cells.

    Science.gov (United States)

    Qi, Dandan; Wang, Qianqian; Li, Hongguang; Zhang, Tao; Lan, Rongfeng; Kwong, Daniel W J; Wong, Wai-Kwok; Wong, Ka-Leung; Li, Shuiming; Lu, Fei

    2015-11-01

    Biological systems have developed an intact network and strategies in response to various environmental pressures such as irradiation, viral invasion and oxidative stress. Therefore, elucidation of the cellular response mechanism toward oxidative stress can contribute to the knowledge of redox regulation. By using a newly developed gadolinium based photodynamic therapy (PDT) agent Gd-N and SILAC quantified proteomic analysis, we observed 485 proteins dysregulated in expression, 106 in phosphorylation and 1050 in oxidation. Interestingly, lysosome was discovered as the main organelle affected by Gd-N induced singlet oxygen, along with the down regulation of a majority of lysosomal acid hydrolases and proton pump complex ATP6V/TCIRG1. Besides, phosphorylation sites with sequence patterns "TP" or "SP" were enriched in dysregulated phosphoproteins. Protein oxidation also shows sequence patterns in target proteins with "M.D" or "KM" taking methionine as the central residue. Oxidized proteins were most enriched in the pathways of Parkinson's disease, an oxidative stress closely related neurodegenerative disease. In conclusion, our study reveals new insights into the cellular mechanism to oxidative stress and may contribute to the discovery of new targets and development of novel PDT agents.

  17. Targeted Genes Sequencing Identified a Novel 15 bp Deletion on GJA8 in a Chinese Family with Autosomal Dominant Congenital Cataracts

    Institute of Scientific and Technical Information of China (English)

    Han-Yi Min; Peng-Peng Qiao; Asan; Zhi-Hui Yan; Hui-Feng Jiang; Ya-Ping Zhu; Hui-Qian Du

    2016-01-01

    Background:Congenital cataract (CC) is the leading cause of visual impairment or blindness in children worldwide.Because of highly genetic and clinical heterogeneity,a molecular diagnosis of the lens disease remains a challenge.Methods:In this study,we tested a three-generation Chinese family with autosomal dominant CCs by targeted sequencing of 45 CC genes on next generation sequencing and evaluated the pathogenicity of the detected mutation by protein structure,pedigree validation,and molecular dynamics (MD) simulation.Results:A novel 15 bp deletion on GJA8 (c.426_440delGCTGGAGGGGACCCT or p.143_147delLEGTL) was detected in the family.The deletion,concemed with an in-frame deletion of 5 amino acid residues in a highly evolutionarily conserved region within the cytoplasmic loop domain of the gap junction channel protein connexin 50 (Cx50),was in full cosegregation with the cataract phenotypes in the family but not found in 1100 control exomes.MD simulation revealed that the introduction of the deletion destabilized the Cx50 gap junction channel,indicating the deletion as a dominant-negative mutation.Conclusions:The above results support the pathogenic role of the 15 bp deletion on GJA8 in the Chinese family and demonstrate targeted genes sequencing as a resolution to molecular diagnosis of CCs.

  18. Development of a robust cell-based high-throughput screening assay to identify targets of HIV-1 viral protein R dimerization

    Directory of Open Access Journals (Sweden)

    Zych C

    2013-05-01

    Full Text Available Courtney Zych,1 Alexander Domling,2 Velpandi Ayyavoo11Department of Infectious Diseases and Microbiology, Graduate School of Public Health, University of Pittsburgh, Pittsburgh, PA, USA; 2Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA, USAAbstract: Targeting protein–protein interactions (PPI is an emerging field in drug discovery. Dimerization and PPI are essential properties of human immunodeficiency virus (HIV-1 proteins, their mediated functions, and virus biology. Additionally, dimerization is required for the functional interaction of HIV-1 proteins with many host cellular components. In this study, a bimolecular fluorescence complementation (BiFC-based screening assay was developed that can quantify changes in dimerization, using HIV-1 viral protein R (Vpr dimerization as a "proof of concept." Results demonstrated that Venus Vpr (generated by BiFC Vpr constructs could be competed off in a dose-dependent manner using untagged, full-length Vpr as a competitor molecule. The change in signal intensity was measured quantitatively through flow cytometry and fluorescence microscopy in a high content screening assay. High content imaging was used to screen a library of small molecules for an effect on Vpr dimerization. Among the tested molecules, a few of the small molecules demonstrate an effect on Vpr dimerization in a dose-dependent manner.Keywords: BiFC, protein–protein interaction, HIV-1 Vpr, dimerization, drug targets

  19. Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets

    DEFF Research Database (Denmark)

    Adriaenssens, Alice E; Svendsen, Berit; Lam, Brian Y H

    2016-01-01

    AIMS/HYPOTHESIS: Intra-islet and gut-islet crosstalk are critical in orchestrating basal and postprandial metabolism. The aim of this study was to identify regulatory proteins and receptors underlying somatostatin secretion though the use of transcriptomic comparison of purified murine alpha, bet...

  20. An in vivo genetic screen in Drosophila identifies the orthologue of human cancer/testis gene SPO11 among a network of targets to inhibit lethal(3)malignant brain tumour growth.

    Science.gov (United States)

    Rossi, Fabrizio; Molnar, Cristina; Hashiyama, Kazuya; Heinen, Jan P; Pampalona, Judit; Llamazares, Salud; Reina, José; Hashiyama, Tomomi; Rai, Madhulika; Pollarolo, Giulia; Fernández-Hernández, Ismael; Gonzalez, Cayetano

    2017-08-01

    Using transgenic RNAi technology, we have screened over 4000 genes to identify targets to inhibit malignant growth caused by the loss of function of lethal(3)malignant brain tumour in Drosophila in vivo We have identified 131 targets, which belong to a wide range of gene ontologies. Most of these target genes are not significantly overexpressed in mbt tumours hence showing that, rather counterintuitively, tumour-linked overexpression is not a good predictor of functional requirement. Moreover, we have found that most of the genes upregulated in mbt tumours remain overexpressed in tumour-suppressed double-mutant conditions, hence revealing that most of the tumour transcriptome signature is not necessarily correlated with malignant growth. One of the identified target genes is meiotic W68 (mei-W68), the Drosophila orthologue of the human cancer/testis gene Sporulation-specific protein 11 (SPO11), the enzyme that catalyses the formation of meiotic double-strand breaks. We show that Drosophila mei-W68/SPO11 drives oncogenesis by causing DNA damage in a somatic tissue, hence providing the first instance in which a SPO11 orthologue is unequivocally shown to have a pro-tumoural role. Altogether, the results from this screen point to the possibility of investigating the function of human cancer relevant genes in a tractable experimental model organism like Drosophila. © 2017 The Authors.

  1. Dataset of the human homologues and orthologues of lipid-metabolic genes identified as DAF-16 targets their roles in lipid and energy metabolism

    Directory of Open Access Journals (Sweden)

    Lavender Yuen-Nam Fan

    2017-04-01

    Full Text Available The data presented in this article are related to the review article entitled ‘Unravelling the role of fatty acid metabolism in cancer through the FOXO3-FOXM1 axis’ (Saavedra-Garcia et al., 2017 [24]. Here, we have matched the DAF-16/FOXO3 downstream genes with their respective human orthologues and reviewed the roles of these targeted genes in FA metabolism. The list of genes listed in this article are precisely selected from literature reviews based on their functions in mammalian FA metabolism. The nematode Caenorhabditis elegans gene orthologues of the genes are obtained from WormBase, the online biological database of C. elegans. This dataset has not been uploaded to a public repository yet.

  2. Transcriptomic profiling of pancreatic alpha, beta and delta cell populations identifies delta cells as a principal target for ghrelin in mouse islets

    DEFF Research Database (Denmark)

    Adriaenssens, Alice E; Svendsen, Berit; Lam, Brian Y H;

    2016-01-01

    and delta cells. METHODS: Sst-Cre mice crossed with fluorescent reporters were used to identify delta cells, while Glu-Venus (with Venus reported under the control of the Glu [also known as Gcg] promoter) mice were used to identify alpha and beta cells. Alpha, beta and delta cells were purified using flow...... cytometry and analysed by RNA sequencing. The role of the ghrelin receptor was validated by imaging delta cell calcium concentrations using islets with delta cell restricted expression of the calcium reporter GCaMP3, and in perfused mouse pancreases. RESULTS: A database was constructed of all genes...... expressed in alpha, beta and delta cells. The gene encoding the ghrelin receptor, Ghsr, was highlighted as being highly expressed and enriched in delta cells. Activation of the ghrelin receptor raised cytosolic calcium levels in primary pancreatic delta cells and enhanced somatostatin secretion in perfused...

  3. Genome-Wide Analysis of In Vivo Binding of the Master Regulator DasR in Streptomyces coelicolor Identifies Novel Non-Canonical Targets

    OpenAIRE

    Świątek-Połatyńska, Magdalena A.; Bucca, Giselda; Laing, Emma; Gubbens, Jacob; Titgemeyer, Fritz; Smith, Colin P; Rigali, Sébastien; van Wezel, Gilles P

    2015-01-01

    Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon o...

  4. Genome-wide profiling of methylation identifies novel targets with aberrant hypermethylation and reduced expression in low-risk myelodysplastic syndromes.

    Science.gov (United States)

    del Rey, M; O'Hagan, K; Dellett, M; Aibar, S; Colyer, H A A; Alonso, M E; Díez-Campelo, M; Armstrong, R N; Sharpe, D J; Gutiérrez, N C; García, J L; De Las Rivas, J; Mills, K I; Hernández-Rivas, J M

    2013-03-01

    Gene expression profiling signatures may be used to classify the subtypes of Myelodysplastic syndrome (MDS) patients. However, there are few reports on the global methylation status in MDS. The integration of genome-wide epigenetic regulatory marks with gene expression levels would provide additional information regarding the biological differences between MDS and healthy controls. Gene expression and methylation status were measured using high-density microarrays. A total of 552 differentially methylated CpG loci were identified as being present in low-risk MDS; hypermethylated genes were more frequent than hypomethylated genes. In addition, mRNA expression profiling identified 1005 genes that significantly differed between low-risk MDS and the control group. Integrative analysis of the epigenetic and expression profiles revealed that 66.7% of the hypermethylated genes were underexpressed in low-risk MDS cases. Gene network analysis revealed molecular mechanisms associated with the low-risk MDS group, including altered apoptosis pathways. The two key apoptotic genes BCL2 and ETS1 were identified as silenced genes. In addition, the immune response and micro RNA biogenesis were affected by the hypermethylation and underexpression of IL27RA and DICER1. Our integrative analysis revealed that aberrant epigenetic regulation is a hallmark of low-risk MDS patients and could have a central role in these diseases.

  5. Targeting aphA : a new high-throughput screening assay identifies compounds that reduce prime virulence factors of Vibrio cholerae.

    Science.gov (United States)

    Bolger, Galina; Roy, Sambit; Zapol'skii, Viktor A; Kaufmann, Dieter E; Schnürch, Michael; Mihovilovic, Marko D; Nandy, Ranjan K; Tegge, Werner

    2016-07-01

    A high-throughput screening (HTS) assay was developed for identifying compounds with inhibitory effect on aphA, one of the key regulators positively controlling Vibrio cholerae pathogenesis. An inhibitory effect on aphA was expected to lead to attenuation in the secretion of the major pathogenicity factors of V. cholerae, cholera toxin and toxin co-regulated pilus. The plasmid construct pAKSB was developed with a kanamycin resistance (KmR) gene under the control of the aphA -like promoter for conferring a KmR phenotype under aphA -expressing conditions. The HTS assay was performed to identify compounds with inhibitory effect on the growth of O139 V. cholerae MO10 carrying the construct pAKSB in growth medium containing Km (30 g ml-1), but not in its absence. Of 20 338 compounds screened, six compounds were identified to inhibit the pAKSB-induced KmR phenotype and these compounds caused transcriptional inhibition of aphA in V. cholerae O139 strain MO10 as well as variant V. cholerae O1 El Tor strain NM06-058. Of the three most active substances, compound 53760866 showed lowest half-maximal cytotoxicity in a eukaryotic cell viability assay and was characterized further. Compound 53760866 caused reduction in cholera toxin secretion and expression of TcpA in vitro. The in vitro virulence attenuation corroborated well in a suckling mouse model in vivo, which showed reduction of colonization by V. cholerae NM06-058 when co-administered with 53760866. The screening method and the compounds may lead to new preventive strategies for cholera by reducing the pathogenicity of V. cholerae .

  6. How Sure Are You That This Is the Man You Saw? Child Witnesses Can Use Confidence Judgments to Identify a Target.

    Science.gov (United States)

    Bruer, Kaila C; Fitzgerald, Ryan J; Price, Heather L; Sauer, James D

    2017-08-17

    We tested whether an alternative lineup procedure designed to minimize problematic influences (e.g., metacognitive development) on decision criteria could be effectively used by children and improve child eyewitness identification performance relative to a standard identification task. Five hundred sixteen children (6- to 13-year-olds) watched a video of a target reading word lists and, the next day, made confidence ratings for each lineup member or standard categorical decisions for 8 lineup members presented sequentially. Two algorithms were applied to classify confidence ratings into categorical decisions and facilitate comparisons across conditions. The classification algorithms produced accuracy rates for the confidence rating procedure that were comparable to the categorical procedure. These findings demonstrate that children can use a ratings-based procedure to discriminate between previously seen and unseen faces. In turn, this invites more nuanced and empirical consideration of ratings-based identification evidence as a probabilistic index of guilt that may attenuate problematic social influences on child witnesses' decision criteria. (PsycINFO Database Record (c) 2017 APA, all rights reserved).

  7. Targeted profiling of arachidonic acid and eicosanoids in rat tissue by UFLC-MS/MS: Application to identify potential markers for rheumatoid arthritis.

    Science.gov (United States)

    Wang, Nannan; Zhao, Xiaoning; Wang, Weihui; Peng, Yan; Bi, Kaishun; Dai, Ronghua

    2017-01-01

    We describe a method for the targeted analysis of bioactive arachidonic acid metabolites through cyclooxygenase (COX) and lipoxygenase (LOX) pathway in knee joint, liver, kidney, spleen and heart using an ultra-fast liquid chromatography-tandem mass (UFLC-MS/MS) method. Method validation was investigated, including linearity, precision, accuracy, matrix effect, extraction recovery and stability for the simultaneous analysis of prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important eicosanoids. The concentrations of the following major eicosanoids were significantly increased in rheumatoid arthritis model rats than in normal ones: 5-HETE, 8-HETE, 12-HETE, 15-HETE, PGF2α, TXB2, 5-HpETE, LTE4, PGE2, PGD2, LTB4. Further multivariate data analysis (partial least square-discriminant analysis) showed COX products (PGs, TXs) were readily distributed towards liver and kidney, LOX products (LTs, HETEs) towards knee joint and spleen, and heart had no characteristic metabolites. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in RA disease states and provides support for use of dual inhibitors of COX and LOX enzymes on RA treatment.

  8. Targeted high-throughput sequencing identifies a TARDBP mutation as a cause of early-onset FTD without motor neuron disease.

    Science.gov (United States)

    Synofzik, Matthis; Born, Christoph; Rominger, Axel; Lummel, Nina; Schöls, Ludger; Biskup, Saskia; Schüle, Cornelius; Grasshoff, Ute; Klopstock, Thomas; Adamczyk, Christopher

    2014-05-01

    Targeted high-throughput sequencing of many amyotrophic lateral sclerosis (ALS) and fronto-temporal dementia (FTD) genes in parallel has the potential to reveal novel ALS- and/or FTD-phenotypes and to provide missing links on the ALS-FTD continuum. For example, although the 43-kDa transactive response DNA binding protein is the major pathologic hallmark linking ALS and FTD, mutations in the gene encoding 43-kDa transactive response DNA binding protein (TARDBP) have been appreciated only as a cause of ALS-phenotypes, but not yet of pure FTD. Thus, the genetic link is not yet well substantiated that TARDBP mutations can cause the full spectrum of the ALS-FTD continuum. High-throughput sequencing of 18 ALS and FTD genes in an index patient presenting with early-onset pure (behavioral) FTD and a positive family history for ALS revealed an established TARDBP mutation, A382T. This finding demonstrates that a TARDPB mutation can cause early-onset pure FTD without evidence for ALS even in advanced FTD disease stages. Moreover, it indicates that TARDPB screening might be considered even in young patients with "pure" neuropsychiatric disturbances and without evidence of neurodegenerative disease in the parental generation.

  9. Multiple antibody targets on herpes B glycoproteins B and D identified by screening sera of infected rhesus macaques with peptide microarrays.

    Directory of Open Access Journals (Sweden)

    Sven-Kevin Hotop

    Full Text Available Herpes B virus (or Herpesvirus simiae or Macacine herpesvirus 1 is endemic in many populations of macaques, both in the wild and in captivity. The virus elicits only mild clinical symptoms (if any in monkeys, but can be transmitted by various routes, most commonly via bites, to humans where it causes viral encephalitis with a high mortality rate. Hence, herpes B constitutes a considerable occupational hazard for animal caretakers, veterinarians and laboratory personnel. Efforts are therefore being made to reduce the risk of zoonotic infection and to improve prognosis after accidental exposure. Among the measures envisaged are serological surveillance of monkey colonies and specific diagnosis of herpes B zoonosis against a background of antibodies recognizing the closely related human herpes simplex virus (HSV. 422 pentadecapeptides covering, in an overlapping fashion, the entire amino acid sequences of herpes B proteins gB and gD were synthesized and immobilized on glass slides. Antibodies present in monkey sera that bind to subsets of the peptide collection were detected by microserological techniques. With 42 different rhesus macaque sera, 114 individual responses to 18 different antibody target regions (ATRs were recorded, 17 of which had not been described earlier. This finding may pave the way for a peptide-based, herpes B specific serological diagnostic test.

  10. Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells.

    Science.gov (United States)

    MacVicar, Thomas D B; Mannack, Lilith V J C; Lees, Robert M; Lane, Jon D

    2015-06-11

    Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy), mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER) and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca2+ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1), as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT) and the IP3-receptors (IP3Rs) as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca2+ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca2+ uniporter (MCU) primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca2+ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca2+ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.

  11. Targeted siRNA Screens Identify ER-to-Mitochondrial Calcium Exchange in Autophagy and Mitophagy Responses in RPE1 Cells

    Directory of Open Access Journals (Sweden)

    Thomas D. B. MacVicar

    2015-06-01

    Full Text Available Autophagy is an important stress response pathway responsible for the removal and recycling of damaged or redundant cytosolic constituents. Mitochondrial damage triggers selective mitochondrial autophagy (mitophagy, mediated by a variety of response factors including the Pink1/Parkin system. Using human retinal pigment epithelial cells stably expressing autophagy and mitophagy reporters, we have conducted parallel screens of regulators of endoplasmic reticulum (ER and mitochondrial morphology and function contributing to starvation-induced autophagy and damage-induced mitophagy. These screens identified the ER chaperone and Ca2+ flux modulator, sigma non-opioid intracellular receptor 1 (SIGMAR1, as a regulator of autophagosome expansion during starvation. Screens also identified phosphatidyl ethanolamine methyl transferase (PEMT and the IP3-receptors (IP3Rs as mediators of Parkin-induced mitophagy. Further experiments suggested that IP3R-mediated transfer of Ca2+ from the ER lumen to the mitochondrial matrix via the mitochondrial Ca2+ uniporter (MCU primes mitochondria for mitophagy. Importantly, recruitment of Parkin to damaged mitochondria did not require IP3R-mediated ER-to-mitochondrial Ca2+ transfer, but mitochondrial clustering downstream of Parkin recruitment was impaired, suggesting involvement of regulators of mitochondrial dynamics and/or transport. Our data suggest that Ca2+ flux between ER and mitochondria at presumed ER/mitochondrial contact sites is needed both for starvation-induced autophagy and for Parkin-mediated mitophagy, further highlighting the importance of inter-organellar communication for effective cellular homeostasis.

  12. Targeted sequencing identifies a novel SH2D1A pathogenic variant in a Chinese family: Carrier screening and prenatal genetic testing

    Science.gov (United States)

    Chen, Yi-Yao; Li, Shu-Yuan; Zhang, Lan-Lan; Shen, Ying-Hua; Chang, Chun-Xin; Xiang, Yu-Qian; Huang, He-Feng; Xu, Chen-Ming

    2017-01-01

    X-linked lymphoproliferative disease type 1 (XLP1) is a rare primary immunodeficiency characterized by a clinical triad consisting of severe EBV-induced hemophagocytic lymphohistiocytosis, B-cell lymphoma, and dysgammaglobulinemia. Mutations in SH2D1A gene have been revealed as the cause of XLP1. In this study, a pregnant woman with recurrence history of birthing immunodeficiency was screened for pathogenic variant because the proband sample was unavailable. We aimed to clarify the genetic diagnosis and provide prenatal testing for the family. Next-generation sequencing (NGS)-based multigene panel was used in carrier screening of the pregnant woman. Variants of immunodeficiency related genes were analyzed and prioritized. Candidate variant was verified by using Sanger sequencing. The possible influence of the identified variant was evaluated through RNA assay. Amniocentesis, karyotyping, and Sanger sequencing were performed for prenatal testing. We identified a novel de novo frameshift SH2D1A pathogenic variant (c.251_255delTTTCA) in the pregnant carrier. Peripheral blood RNA assay indicated that the mutant transcript could escape nonsense-mediated mRNA decay (NMD) and might encode a C-terminal truncated protein. Information of the variant led to success prenatal diagnosis of the fetus. In conclusion, our study clarified the genetic diagnosis and altered disease prevention for a pregnant carrier of XLP1. PMID:28231257

  13. Disparities in mammographic screening for Asian women in California: a cross-sectional analysis to identify meaningful groups for targeted intervention

    Directory of Open Access Journals (Sweden)

    Keegan Theresa HM

    2007-10-01

    Full Text Available Abstract Background Breast cancer is the most commonly diagnosed cancer among the rapidly growing population of Asian Americans; it is also the most common cause of cancer mortality among Filipinas. Asian women continue to have lower rates of mammographic screening than women of most other racial/ethnic groups. While prior studies have described the effects of sociodemographic and other characteristics of women on non-adherence to screening guidelines, they have not identified the distinct segments of the population who remain at highest risk of not being screened. Methods To better describe characteristics of Asian women associated with not having a mammogram in the last two years, we applied recursive partitioning to population-based data (N = 1521 from the 2001 California Health Interview Survey (CHIS, for seven racial/ethnic groups of interest: Chinese, Japanese, Filipino, Korean, South Asian, Vietnamese, and all Asians combined. Results We identified two major subgroups of Asian women who reported not having a mammogram in the past two years and therefore, did not follow mammography screening recommendations: 1 women who have never had a pap exam to screen for cervical cancer (68% had no mammogram, and 2 women who have had a pap exam, but have no women's health issues (osteoporosis, using menopausal hormone therapies, and/or hysterectomy nor a usual source of care (62% had no mammogram. Only 19% of Asian women who have had pap screening and have women's health issues did not have a mammogram in the past two years. In virtually all ethnic subgroups, having had pap or colorectal screening were the strongest delineators of mammography usage. Other characteristics of women least likely to have had a mammogram included: Chinese non-U.S. citizens or citizens without usual source of health care, Filipinas with no health insurance, Koreans without women's health issues and public or no health insurance, South Asians less than age 50 who were

  14. Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets.

    Science.gov (United States)

    Alfaro, Joshua F; Gong, Cheng-Xin; Monroe, Matthew E; Aldrich, Joshua T; Clauss, Therese R W; Purvine, Samuel O; Wang, Zihao; Camp, David G; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W; Hunt, Donald F; Yang, Feng; Smith, Richard D

    2012-05-01

    O-linked N-acetylglucosamine (O-GlcNAc) is a reversible posttranslational modification of Ser and Thr residues on cytosolic and nuclear proteins of higher eukaryotes catalyzed by O-GlcNAc transferase (OGT). O-GlcNAc has recently been found on Notch1 extracellular domain catalyzed by EGF domain-specific OGT. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing ∼100 μg of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these, 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located proximal to known phosphorylation sites. These findings support the proposed regulatory cross-talk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-β-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, expanding the evidence for extracellular O-GlcNAcylation by the EGF domain-specific OGT. We also report a GlcNAc-β-1,3-Fuc-α-1-O-Thr modification on the EGF-like repeat of the versican core protein, a proposed substrate of Fringe β-1,3-N-acetylglucosaminyltransferases.

  15. Tandem Mass Spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets

    Energy Technology Data Exchange (ETDEWEB)

    Alfaro, Joshua F.; Gong, Cheng-Xin; Monroe, Matthew E.; Aldrich, Joshua T.; Clauss, Therese RW; Purvine, Samuel O.; Wang, Zihao; Camp, David G.; Shabanowitz, Jeffrey; Stanley, Pamela; Hart, Gerald W.; Hunt, Donald F.; Yang, Feng; Smith, Richard D.

    2012-05-08

    O-Linked N-Acetylglucosamine (O-GlcNAc) is a reversible post-translational modification of Ser and Thr residues on cytosolic and nuclear proteins found in all higher eukaryotes. Aberrant O-GlcNAc modification of brain proteins has been linked to Alzheimer's disease (AD). However, understanding specific functions of O-GlcNAcylation in AD has been impeded by the difficulty in characterization of O-GlcNAc sites on proteins. In this study, we modified a chemical/enzymatic photochemical cleavage approach for enriching O-GlcNAcylated peptides in samples containing {approx}100 {micro}g of tryptic peptides from mouse cerebrocortical brain tissue. A total of 274 O-GlcNAcylated proteins were identified. Of these 168 were not previously known to be modified by O-GlcNAc. Overall, 458 O-GlcNAc sites on Ser and Thr residues in 195 proteins were identified. Many of the modified residues are either known phosphorylation sites or located in close proximity to known phosphorylation sites. These findings support the proposed regulatory crosstalk between O-GlcNAcylation and phosphorylation. This study produced the most comprehensive O-GlcNAc proteome of mammalian brain tissue with both protein identification and O-GlcNAc site assignment. Interestingly, we observed O-{beta}-GlcNAc on EGF-like repeats in the extracellular domains of five membrane proteins, thus representing the first evidence for extracellular O-GlcNAcylation in mammalian systems by the ER-resident O-GlcNAc transferase (EOGT). We also report a GlcNAc-{beta}-1,3-Fuc-{alpha}-1-O-Thr modification on the EGF-like repeat of the Versican core protein, a novel substrate of Fringe {beta}1,3-N-acetylglucosaminyltransferases.

  16. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

    Science.gov (United States)

    Imaduwage, Kasun P.; Go, Eden P.; Zhu, Zhikai; Desaire, Heather

    2016-09-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

  17. HAMS: High-Affinity Mass Spectrometry Screening. A High-Throughput Screening Method for Identifying the Tightest-Binding Lead Compounds for Target Proteins with No False Positive Identifications

    Science.gov (United States)

    Imaduwage, Kasun P.; Go, Eden P.; Zhu, Zhikai; Desaire, Heather

    2016-11-01

    A major challenge in drug discovery is the identification of high affinity lead compounds that bind a particular target protein; these leads are typically identified by high throughput screens. Mass spectrometry has become a detection method of choice in drug screening assays because the target and the ligand need not be modified. Label-free assays are advantageous because they can be developed more rapidly than assays requiring labels, and they eliminate the risk of the label interfering with the binding event. However, in commonly used MS-based screening methods, detection of false positives is a major challenge. Here, we describe a detection strategy designed to eliminate false positives. In this approach, the protein and the ligands are incubated together, and the non-binders are separated for detection. Hits (protein binders) are not detectable by MS after incubation with the protein, but readily identifiable by MS when the target protein is not present in the incubation media. The assay was demonstrated using three different proteins and hundreds of non-inhibitors; no false positive hits were identified in any experiment. The assay can be tuned to select for ligands of a particular binding affinity by varying the quantity of protein used and the immobilization method. As examples, the method selectively detected inhibitors that have Ki values of 0.2 μM, 50 pM, and 700 pM. These findings demonstrate that the approach described here compares favorably with traditional MS-based screening methods.

  18. The recently identified P2Y-like receptor GPR17 is a sensor of brain damage and a new target for brain repair.

    Directory of Open Access Journals (Sweden)

    Davide Lecca

    promoted the expression of myelin basic protein, confirming progression toward mature oligodendrocytes. Thus, GPR17 may act as a "sensor" that is activated upon brain injury on several embryonically distinct cell types, and may play a key role in both inducing neuronal death inside the ischemic core and in orchestrating the local remodeling/repair response. Specifically, we suggest GPR17 as a novel target for therapeutic manipulation to foster repair of demyelinating wounds, the types of lesions that also occur in patients with multiple sclerosis.

  19. Genome-wide analysis of in vivo binding of the master regulator DasR in Streptomyces coelicolor identifies novel non-canonical targets.

    Science.gov (United States)

    Świątek-Połatyńska, Magdalena A; Bucca, Giselda; Laing, Emma; Gubbens, Jacob; Titgemeyer, Fritz; Smith, Colin P; Rigali, Sébastien; van Wezel, Gilles P

    2015-01-01

    Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon of DasR extends well beyond what was previously predicted and includes a large number of genes with functions far removed from N-acetylglucosamine metabolism, such as genes for small RNAs and DNA transposases. Conversely, the DasR regulon during vegetative growth largely correlates to the presence of canonical DasR-responsive elements. The changes in DasR binding in vivo following N-acetylglucosamine induction were studied in detail and a possible molecular mechanism by which the influence of DasR is extended is discussed. Discussion of DasR binding was further informed by a parallel transcriptome analysis of the respective cultures. Evidence is provided that DasR binds directly to the promoters of all genes encoding pathway-specific regulators of antibiotic production in S. coelicolor, thereby providing an exquisitely simple link between nutritional control and secondary metabolism.

  20. Genome-wide analysis of in vivo binding of the master regulator DasR in Streptomyces coelicolor identifies novel non-canonical targets.

    Directory of Open Access Journals (Sweden)

    Magdalena A Świątek-Połatyńska

    Full Text Available Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon of DasR extends well beyond what was previously predicted and includes a large number of genes with functions far removed from N-acetylglucosamine metabolism, such as genes for small RNAs and DNA transposases. Conversely, the DasR regulon during vegetative growth largely correlates to the presence of canonical DasR-responsive elements. The changes in DasR binding in vivo following N-acetylglucosamine induction were studied in detail and a possible molecular mechanism by which the influence of DasR is extended is discussed. Discussion of DasR binding was further informed by a parallel transcriptome analysis of the respective cultures. Evidence is provided that DasR binds directly to the promoters of all genes encoding pathway-specific regulators of antibiotic production in S. coelicolor, thereby providing an exquisitely simple link between nutritional control and secondary metabolism.

  1. Novel PSEN1 mutations (H214N and R220P) associated with familial Alzheimer's disease identified by targeted exome sequencing.

    Science.gov (United States)

    Piccoli, Elena; Rossi, Giacomina; Rossi, Tommaso; Pelliccioni, Giuseppe; D'Amato, Ilaria; Tagliavini, Fabrizio; Di Fede, Giuseppe

    2016-04-01

    Autosomal dominant Alzheimer's disease (AD) is caused by mutations in amyloid precursor protein, presenilin 1 (PSEN1), and presenilin 2 genes and is mostly associated with early-onset form of AD (EOAD), whereas very few mutations were also found in late-onset AD (LOAD) cases. Because of the clinical overlapping between AD and other degenerative dementias such as frontotemporal dementias, a wide-spectrum genetic analysis should be envisaged in the differential diagnosis of this group of disorders. We used next-generation sequencing techniques to analyze 10 genes involved in dementia on a cohort of 20 EOAD and 20 LOAD cases. We found 5 rare coding variants (frequency <1%). PSEN1 H214N mutation, identified in a case of familial EOAD and PSEN1 R220P, found in a case of familial LOAD, are predicted to be pathogenic. These findings confirm the contribution of PSEN1 genetic variants also to LOAD, underlining the need of extending the genetic screening of presenilin mutations to LOAD patients. Two variants in microtubule-associated protein tau and 1 in progranulin appeared to be benign polymorphisms, showing no major contribution of these genes to AD.

  2. Genome-Wide Analysis of In Vivo Binding of the Master Regulator DasR in Streptomyces coelicolor Identifies Novel Non-Canonical Targets

    Science.gov (United States)

    Świątek-Połatyńska, Magdalena A.; Bucca, Giselda; Laing, Emma; Gubbens, Jacob; Titgemeyer, Fritz; Smith, Colin P.; Rigali, Sébastien; van Wezel, Gilles P.

    2015-01-01

    Streptomycetes produce a wealth of natural products, including over half of all known antibiotics. It was previously demonstrated that N-acetylglucosamine and secondary metabolism are closely entwined in streptomycetes. Here we show that DNA recognition by the N-acetylglucosamine-responsive regulator DasR is growth-phase dependent, and that DasR can bind to sites in the S. coelicolor genome that have no obvious resemblance to previously identified DasR-responsive elements. Thus, the regulon of DasR extends well beyond what was previously predicted and includes a large number of genes with functions far removed from N-acetylglucosamine metabolism, such as genes for small RNAs and DNA transposases. Conversely, the DasR regulon during vegetative growth largely correlates to the presence of canonical DasR-responsive elements. The changes in DasR binding in vivo following N-acetylglucosamine induction were studied in detail and a possible molecular mechanism by which the influence of DasR is extended is discussed. Discussion of DasR binding was further informed by a parallel transcriptome analysis of the respective cultures. Evidence is provided that DasR binds directly to the promoters of all genes encoding pathway-specific regulators of antibiotic production in S. coelicolor, thereby providing an exquisitely simple link between nutritional control and secondary metabolism. PMID:25875084

  3. Highly Selective Targeting of Hepatic Stellate Cells for Liver Fibrosis Treatment Using a d-Enantiomeric Peptide Ligand of Fn14 Identified by Mirror-Image mRNA Display.

    Science.gov (United States)

    Huang, Luying; Xie, Jing; Bi, Qiuyan; Li, Zhuoxuan; Liu, Sha; Shen, Qing; Li, Chong

    2017-05-01

    Although liver fibrosis is a major public health issue, there is still no effective drug therapy in the clinic. Fibroblast growth factor-inducible 14 (Fn14), a membrane receptor highly specifically expressed in activated hepatic stellate cells (HSCs), is the key driver of liver fibrosis, and thus, it has a great potential as a novel target for the development of effective treatment. Here, we identified a d-enantiomeric peptide ligand of Fn14 through mirror-image mRNA display. This included the chemical synthesis of a d-enantiomer of the target protein (extracellular domain of Fn14), identification of an l-peptide ligand of d-Fn14 using a constructed mRNA peptide library, and identification of a d-enantiomer of the l-peptide, which is a ligand of the natural Fn14 for reasons of symmetry. The obtained d-peptide ligand showed strong binding to Fn14 while maintaining high proteolytic resistance. As a targeting moiety, this d-peptide successfully mediated high selectivity of activated HSCs for liposomal vehicles compared to that of other major cell types in the liver and significantly enhanced the accumulation of liposomes in the liver fibrosis region of a carbon tetrachloride-induced mouse model. Moreover, in combination with curcumin as an encapsulated load, a liposomal formulation conjugated with this d-peptide showed powerful inhibition of the proliferation of activated HSCs and reduced the liver fibrosis to a significant extent in vivo. This Fn14-targeting strategy may represent a promising approach to targeted drug delivery for liver fibrosis treatment. Meanwhile, the mirror-image mRNA display can provide a new arsenal for the development of d-peptide-based therapeutics against a variety of human diseases.

  4. Utilizing the Dog Genome in the Search for Novel Candidate Genes Involved in Glioma Development-Genome Wide Association Mapping followed by Targeted Massive Parallel Sequencing Identifies a Strongly Associated Locus.

    Directory of Open Access Journals (Sweden)

    Katarina Truvé

    2016-05-01

    Full Text Available Gliomas are the most common form of malignant primary brain tumors in humans and second most common in dogs, occurring with similar frequencies in both species. Dogs are valuable spontaneous models of human complex diseases including cancers and may provide insight into disease susceptibility and oncogenesis. Several brachycephalic breeds such as Boxer, Bulldog and Boston Terrier have an elevated risk of developing glioma, but others, including Pug and Pekingese, are not at higher risk. To identify glioma-associated genetic susceptibility factors, an across-breed genome-wide association study (GWAS was performed on 39 dog glioma cases and 141 controls from 25 dog breeds, identifying a genome-wide significant locus on canine chromosome (CFA 26 (p = 2.8 x 10-8. Targeted re-sequencing of the 3.4 Mb candidate region was performed, followed by genotyping of the 56 SNVs that best fit the association pattern between the re-sequenced cases and controls. We identified three candidate genes that were highly associated with glioma susceptibility: CAMKK2, P2RX7 and DENR. CAMKK2 showed reduced expression in both canine and human brain tumors, and a non-synonymous variant in P2RX7, previously demonstrated to have a 50% decrease in receptor function, was also associated with disease. Thus, one or more of these genes appear to affect glioma susceptibility.

  5. Utilizing the Dog Genome in the Search for Novel Candidate Genes Involved in Glioma Development—Genome Wide Association Mapping followed by Targeted Massive Parallel Sequencing Identifies a Strongly Associated Locus

    Science.gov (United States)

    Dickinson, Peter; Xiong, Anqi; York, Daniel; Jayashankar, Kartika; Pielberg, Gerli; Koltookian, Michele; Murén, Eva; Fuxelius, Hans-Henrik; Weishaupt, Holger; Andersson, Göran; Hedhammar, Åke; Bongcam-Rudloff, Erik; Forsberg-Nilsson, Karin

    2016-01-01

    Gliomas are the most common form of malignant primary brain tumors in humans and second most common in dogs, occurring with similar frequencies in both species. Dogs are valuable spontaneous models of human complex diseases including cancers and may provide insight into disease susceptibility and oncogenesis. Several brachycephalic breeds such as Boxer, Bulldog and Boston Terrier have an elevated risk of developing glioma, but others, including Pug and Pekingese, are not at higher risk. To identify glioma-associated genetic susceptibility factors, an across-breed genome-wide association study (GWAS) was performed on 39 dog glioma cases and 141 controls from 25 dog breeds, identifying a genome-wide significant locus on canine chromosome (CFA) 26 (p = 2.8 x 10−8). Targeted re-sequencing of the 3.4 Mb candidate region was performed, followed by genotyping of the 56 SNVs that best fit the association pattern between the re-sequenced cases and controls. We identified three candidate genes that were highly associated with glioma susceptibility: CAMKK2, P2RX7 and DENR. CAMKK2 showed reduced expression in both canine and human brain tumors, and a non-synonymous variant in P2RX7, previously demonstrated to have a 50% decrease in receptor function, was also associated with disease. Thus, one or more of these genes appear to affect glioma susceptibility. PMID:27171399

  6. Targeted re-sequencing identified rs3106189 at the 5' UTR of TAPBP and rs1052918 at the 3' UTR of TCF3 to be associated with the overall survival of colorectal cancer patients.

    Directory of Open Access Journals (Sweden)

    Jiaofang Shao

    Full Text Available Recent studies have demonstrated the power of deep re-sequencing of the whole genome or exome in understanding cancer genomes. However, targeted capture of selected genomic whole gene-body regions, rather than the whole exome, have several advantages: 1 the genes can be selected based on biology or a hypothesis; 2 mutations in promoter and intronic regions, which have important regulatory roles, can be investigated; and 3 less expensive than whole genome or whole exome sequencing. Therefore, we designed custom high-density oligonucleotide microarrays (NimbleGen Inc. to capture approximately 1.7 Mb target regions comprising the genomic regions of 28 genes related to colorectal cancer including genes belonging to the WNT signaling pathway, as well as important transcription factors or colon-specific genes that are over expressed in colorectal cancer (CRC. The 1.7 Mb targeted regions were sequenced with a coverage ranged from 32× to 45× for the 28 genes. We identified a total of 2342 sequence variations in the CRC and corresponding adjacent normal tissues. Among them, 738 were novel sequence variations based on comparisons with the SNP database (dbSNP135. We validated 56 of 66 SNPs in a separate cohort of 30 CRC tissues using Sequenom MassARRAY iPLEX Platform, suggesting a validation rate of at least 85% (56/66. We found 15 missense mutations among the exonic variations, 21 synonymous SNPs that were predicted to change the exonic splicing motifs, 31 UTR SNPs that were predicted to occur at the transcription factor binding sites, 20 intronic SNPs located near the splicing sites, 43 SNPs in conserved transcription factor binding sites and 32 in CpG islands. Finally, we determined that rs3106189, localized to the 5' UTR of antigen presenting tapasin binding protein (TAPBP, and rs1052918, localized to the 3' UTR of transcription factor 3 (TCF3, were associated with overall survival of CRC patients.

  7. Resolution of the cellular proteome of the nucleocapsid protein from a highly pathogenic isolate of porcine reproductive and respiratory syndrome virus identifies PA