Systematic interpretation of molecular beacon polymerase chain reaction for identifying rpoB mutations in Mycobacterium tuberculosis isolates with mixed resistant and susceptible bacteria

Detection of multidrug-resistant tuberculosis (MDR-TB), a frequent cause of treatment failure, takes 2 or more weeks to identify by culture. RIF-resistance is a hallmark of MDR-TB, and detection of mutations in the rpoB gene of Mycobacterium tuberculosis using molecular beacon probes with real-time quantitative polymerase chain reaction (qPCR) is a novel approach that takes ≤2 days. However, qPCR identification of resistant isolates, particularly for isolates with mixed RIF-susceptible and RIF-resistant bacteria, is reader dependent and limits its clinical use. The aim of this study was to develop an objective, reader-independent method to define rpoB mutants using beacon qPCR. This would facilitate the transition from a research protocol to the clinical setting, where high-throughput methods with objective interpretation are required. For this, DNAs from 107 M. tuberculosis clinical isolates with known susceptibility to RIF by culture-based methods were obtained from 2 regions where isolates have not previously been subjected to evaluation using molecular beacon qPCR: the Texas–Mexico border and Colombia. Using coded DNA specimens, mutations within an 81-bp hot spot region of rpoB were established by qPCR with 5 beacons spanning this region. Visual and mathematical approaches were used to establish whether the qPCR cycle threshold of the experimental isolate was significantly higher (mutant) compared to a reference wild-type isolate. Visual classification of the beacon qPCR required reader training for strains with a mixture of RIF-susceptible and RIF-resistant bacteria. Only then had the visual interpretation by an experienced reader had 100% sensitivity and 94.6% specificity versus RIF-resistance by culture phenotype and 98.1% sensitivity and 100% specificity versus mutations based on DNA sequence. The mathematical approach was 98% sensitive and 94.5% specific versus culture and 96.2% sensitive and 100% specific versus DNA sequence. Our findings indicate the mathematical approach has advantages over the visual reading, in that it uses a Microsoft Excel template to eliminate reader bias or inexperience, and allows objective interpretation from high-throughput analyses even in the presence of a mixture of RIF-resistant and RIF-susceptible isolates without the need for reader training.^

Quantitative trait loci and metabolic pathways: genetic control of the concentration of maysin, a corn earworm resistance factor, in maize silks.

Interpretation of quantitative trait locus (QTL) studies of agronomic traits is limited by lack of knowledge of biochemical pathways leading to trait expression. To more fully elucidate the biological significance of detected QTL, we chose a trait that is the product of a well-characterized pathway, namely the concentration of maysin, a C-glycosyl flavone, in silks of maize, Zea mays L. Maysin is a host-plant resistance factor against the corn earworm, Helicoverpa zea (Boddie). We determined silk maysin concentrations and restriction fragment length polymorphism genotypes at flavonoid pathway loci or linked markers for 285 F2 plants derived from the cross of lines GT114 and GT119. Single-factor analysis of variance indicated that the p1 region on chromosome 1 accounted for 58.0% of the phenotypic variance and showed additive gene action. The p1 locus is a transcription activator for portions of the flavonoid pathway. A second QTL, represented by marker umc 105a near the brown pericarp1 locus on chromosome 9, accounted for 10.8% of the variance. Gene action of this region was dominant for low maysin, but was only expressed in the presence of a functional p1 allele. The model explaining the greatest proportion of phenotypic variance (75.9%) included p1, umc105a, umc166b (chromosome 1), r1 (chromosome 10), and two epistatic interaction terms, p1 x umc105a and p1 x r1. Our results provide evidence that regulatory loci have a central role and that there is a complex interplay among different branches of the flavonoid pathway in the expression of this trait.

Retention, adherence and compliance: Important considerations for home- and group-based resistance training programs for older adults

Reports on the efficacy of physical activity intervention trials usually only include discussion of the primary outcomes. However, assessing factors such as participant retention, adherence and compliance can assist in the accurate interpretation of the overall impact of a program in terms of reach and appeal. A quasi-randomised trial was carried out to assess and compare retention and adherence rates, and compliance with, a twice weekly resistance training program provided either individually at home or in a group format. Retirement villages (n=6) were assigned to either 'Have A Try' (HAT, home-based) or 'Come Have A Try' (CHAT, group-based); both programs included nine strength and two balance exercises. The program involved a 20-week Intervention Phase a 24-week Maintenance Phase and a 20-week On-going Maintenance Phase. One hundred and nineteen participants (mean age 80 +/- 6 years) were recruited (HAT = 38, CHAT = 81). There was no difference in retention rates at the end of the Intervention Phase, but significantly more HAT than CHAT participants had dropped out of the study (p < 0.01) after the Maintenance Phase and the On-going Maintenance Phase. During the Intervention Phase, over half the HAT and CHAT participants completed >= 75% of the prescribed activity sessions, but adherence was significantly greater in CHAT than HAT during the Maintenance Phase (p < 0.01). Participants in CHAT were significantly more compliant than HAT participants (p < 0.05). Both home- and group-based formats were successful over the short-term, but, in retirement villages, the group program had better adherence and compliance in the longer-term. (c) 2006 Sports Medicine Australia. Published by Elsevier Ltd. All rights reserved.

Boundary maintenance in Qatar: Interpretation of domination, segregation and subordination

This study explores the interaction of expatriates in Qatar and their perception of their subordination. The study design included participant observation in an all female University and University housing as well as interviews with Qatari government agencies and ministries, expatriate embassies and expatriates. Semi-structured interviews were conducted across seven expatriate groups: domestic workers, unskilled laborers, semiskilled, professionals, housewives, second-generation expatriates with host country other than Qatar, second-generation expatriates with host country Qatar, and Gulf Cooperation Council citizens. Forty-two subjects completed the interview schedule while 87 interviews were incomplete. ^ Physical control of expatriates occurs through the Gulf practice of sponsorship (The Kafeel System), and local cultural and Islamic related controls intertwined with the Arab Code of honor. Interviews and observations revealed rankings of Arabs and foreigners which emphasize Qatari superiority such as tribal identity, moral ranking of female groups by dress, legal protection and power, sexual consideration and desexualization and salaries and job opportunities based on nationality and ethnicity. Individuals who desire to transcend boundaries into the Qatari realm through citizenship or marriage view Qataris as possessing the “image of the unlimited good” and have acquired Qatari social and cultural capital. Members from all expatriate groups engaged in various forms of resistance to labor and gender domination which ranged from forms of “exit,” expressing a hidden transcript in the privacy of their own group, disguised resistance in public, and occasionally, direct confrontation with the Qatari. Although the legal arena created the appearance that worker's needs were being addressed, laborers engaged in forms of “exit” to escape their oppression. Omani students in the hostel disguised their resistance by spreading gossip, nick-naming homosexual Qatari students at the University, acting out a skit depicting their exclusion from Qatari privilege, spreading rumors of impending freedom, and singing songs of despair in the courtyard. Other sites of resistance were expatriate embassies, the road, the newspaper and technology. This study emphasizes that blaming oppression of the expatriate worker on globalization is a simplistic view of oppression in the Gulf, and ignores complex issues within Qatari society and other Gulf States. Sponsorship, servitude, and gender segregation intersect in Qatar to create a system of segregation and domination of expatriates. ^

Resistance and resilience to droughts : hydropedological controls on catchment storage and run-off response

Acknowledgements The authors would like to thank Jonathan Dick, Maria Blumstock, Claire Tunaley and Jason Lessels for assistance with the field work and Audrey Innes for lab sample preparation. Climatic data were provided by Iain Malcolm and Marine Scotland Fisheries at the Freshwater Lab, Pitlochry. Additional precipitation and temperature data were provided by the UK Meteorological Office and the British Atmospheric Data Centre (BADC). We are grateful for the careful and constructive comments of two anonymous reviewers that helped to improve an earlier version of this manuscript. The European Research Council ERC (project GA 335910) is thanked for funding.

Resistance and resilience to droughts : hydropedological controls on catchment storage and run-off response

Acknowledgements The authors would like to thank Jonathan Dick, Maria Blumstock, Claire Tunaley and Jason Lessels for assistance with the field work and Audrey Innes for lab sample preparation. Climatic data were provided by Iain Malcolm and Marine Scotland Fisheries at the Freshwater Lab, Pitlochry. Additional precipitation and temperature data were provided by the UK Meteorological Office and the British Atmospheric Data Centre (BADC). We are grateful for the careful and constructive comments of two anonymous reviewers that helped to improve an earlier version of this manuscript. The European Research Council ERC (project GA 335910) is thanked for funding.

Shadows of militarism: an ethnography of trauma and resistance among soldiers and veterans in post-9/11 USA.

This thesis critically examines the military disciplining of trauma through a detailed ethnographic study of post-9/11 lower-enlisted soldiers and veterans in the U.S. who have links to a national movement of resistance to, and healing from, militarism. Drawing on 12 months of ethnographic fieldwork at two G.I. coffeehouses and with the post-9/11 veteran anti-militarism movement in U.S., it analyses the journey of joining the military, becoming a soldier, leaving the military and veteran identities. It explores militarism and military power as a cultural process which reproduces and conceals itself within normative conceptions of the everyday, and military trauma as a site of contested power and resistance. In doing so, this research addresses an urgent need to critically engage with military trauma as a means to challenge normalised discourses of militarism. This research reveals a disjuncture between the imagined and lived reality of military identities in the post-9/11 era. It explores the politics of recognition of veterans’ public and private lives, their contested identities, and their constrained relationship to the state. It argues that veterans are silenced and their identities reduced to symbolic tools in a public military imaginary which constructs military trauma into politically manageable categories, while disciplining and silencing the nation from critically examining war and militarism. In this way, this thesis argues that veterans serve a vital function in U.S. society by absorbing and containing the violence of the state, which then becomes unspeakable, unhearable, and inescapable. This thesis shows how a small number of soldiers and veterans are pushing back against this narrative. In sum, this thesis seeks to challenge the disciplinary effects of militarism upon trauma and support veteran voices to speak their own truths.

Ikkε Is Key To Induction Of Insulin Resistance In The Hypothalamus, And Its Inhibition Reverses Obesity.

IKK epsilon (IKKε) is induced by the activation of nuclear factor-κB (NF-κB). Whole-body IKKε knockout mice on a high-fat diet (HFD) were protected from insulin resistance and showed altered energy balance. We demonstrate that IKKε is expressed in neurons and is upregulated in the hypothalamus of obese mice, contributing to insulin and leptin resistance. Blocking IKKε in the hypothalamus of obese mice with CAYMAN10576 or small interfering RNA decreased NF-κB activation in this tissue, relieving the inflammatory environment. Inhibition of IKKε activity, but not TBK1, reduced IRS-1(Ser307) phosphorylation and insulin and leptin resistance by an improvement of the IR/IRS-1/Akt and JAK2/STAT3 pathways in the hypothalamus. These improvements were independent of body weight and food intake. Increased insulin and leptin action/signaling in the hypothalamus may contribute to a decrease in adiposity and hypophagia and an enhancement of energy expenditure accompanied by lower NPY and increased POMC mRNA levels. Improvement of hypothalamic insulin action decreases fasting glycemia, glycemia after pyruvate injection, and PEPCK protein expression in the liver of HFD-fed and db/db mice, suggesting a reduction in hepatic glucose production. We suggest that IKKε may be a key inflammatory mediator in the hypothalamus of obese mice, and its hypothalamic inhibition improves energy and glucose metabolism.

Neck Circumference As A New Anthropometric Indicator For Prediction Of Insulin Resistance And Components Of Metabolic Syndrome In Adolescents: Brazilian Metabolic Syndrome Study.

To evaluate the correlation between neck circumference and insulin resistance and components of metabolic syndrome in adolescents with different adiposity levels and pubertal stages, as well as to determine the usefulness of neck circumference to predict insulin resistance in adolescents. Cross-sectional study with 388 adolescents of both genders from ten to 19 years old. The adolescents underwent anthropometric and body composition assessment, including neck and waist circumferences, and biochemical evaluation. The pubertal stage was obtained by self-assessment, and the blood pressure, by auscultation. Insulin resistance was evaluated by the Homeostasis Model Assessment-Insulin Resistance. The correlation between two variables was evaluated by partial correlation coefficient adjusted for the percentage of body fat and pubertal stage. The performance of neck circumference to identify insulin resistance was tested by Receiver Operating Characteristic Curve. After the adjustment for percentage body fat and pubertal stage, neck circumference correlated with waist circumference, blood pressure, triglycerides and markers of insulin resistance in both genders. The results showed that the neck circumference is a useful tool for the detection of insulin resistance and changes in the indicators of metabolic syndrome in adolescents. The easiness of application and low cost of this measure may allow its use in Public Health services.

Undulatory Physical Resistance Training Program Increases Maximal Strength In Elderly Type 2 Diabetics.

To investigate the effects of a specific protocol of undulatory physical resistance training on maximal strength gains in elderly type 2 diabetics. The study included 48 subjects, aged between 60 and 85 years, of both genders. They were divided into two groups: Untrained Diabetic Elderly (n=19) with those who were not subjected to physical training and Trained Diabetic Elderly (n=29), with those who were subjected to undulatory physical resistance training. The participants were evaluated with several types of resistance training's equipment before and after training protocol, by test of one maximal repetition. The subjects were trained on undulatory resistance three times per week for a period of 16 weeks. The overload used in undulatory resistance training was equivalent to 50% of one maximal repetition and 70% of one maximal repetition, alternating weekly. Statistical analysis revealed significant differences (p<0.05) between pre-test and post-test over a period of 16 weeks. The average gains in strength were 43.20% (knee extension), 65.00% (knee flexion), 27.80% (supine sitting machine), 31.00% (rowing sitting), 43.90% (biceps pulley), and 21.10% (triceps pulley). Undulatory resistance training used with weekly different overloads was effective to provide significant gains in maximum strength in elderly type 2 diabetic individuals.

Urothelial Carcinogen Resistance Driven By Stronger Toll-like Receptor 2 (tlr2) And Uroplakin Iii (up Iii) Defense Mechanisms: A New Model.

The objective of the study was to illustrate the applicability and significance of the novel Lewis urothelial cancer model compared to the classic Fisher 344. Fischer 344 and Lewis females rats, 7 weeks old, were intravesical instilled N-methyl-N-nitrosourea 1.5 mg/kg every other week for a total of four doses. After 15 weeks, animals were sacrificed and bladders analyzed: histopathology (tumor grade and stage), immunohistochemistry (apoptotic and proliferative indices) and blotting (Toll-like receptor 2-TLR2, Uroplakin III-UP III and C-Myc). Control groups received placebo. There were macroscopic neoplastic lesions in 20 % of Lewis strain and 70 % of Fischer 344 strain. Lewis showed hyperplasia in 50 % of animals, normal bladders in 50 %. All Fischer 344 had lesions, 20 % papillary hyperplasia, 30 % dysplasia, 40 % neoplasia and 10 % squamous metaplasia. Proliferative and apoptotic indices were significantly lower in the Lewis strain (p < 0.01). The TLR2 and UP III protein levels were significantly higher in Lewis compared to Fischer 344 strain (70.8 and 46.5 % vs. 49.5 and 16.9 %, respectively). In contrast, C-Myc protein levels were significantly higher in Fischer 344 (22.5 %) compared to Lewis strain (13.7 %). The innovative Lewis carcinogen resistance urothelial model represents a new strategy for translational research. Preservation of TLR2 and UP III defense mechanisms might drive diverse urothelial phenotypes during carcinogenesis in differently susceptible individuals.

Mechanisms Of Insulin Resistance In The Amygdala: Influences On Food Intake.

Obesity is increasing worldwide and is triggered, at least in part, by enhanced caloric intake. Food intake is regulated by a complex mechanism involving the hypothalamus and hindbrain circuitries. However, evidences have showing that reward systems are also important in regulating feeding behavior. In this context, amygdala is considered a key extra-hypothalamic area regulating feeding behavior in human beings and rodents. This review focuses on the regulation of food intake by amygdala and the mechanisms of insulin resistance in this brain area. Similar to the hypothalamus the anorexigenic effect of insulin is mediated via PI3K (phosphoinositide 3-kinase)/Akt (protein kinase B) pathway in the amygdala. Insulin decreases NPY (neuropeptide Y) and increases oxytocin mRNA levels in the amygdala. High fat diet and saturated fatty acids induce inflammation, ER (endoplasmic reticulum) stress and the activation of serine kinases such as PKCθ (protein kinase C theta), JNK (c-Jun N-terminal kinase) and IKKβ (inhibitor of nuclear factor kappa-B kinase beta) in the amygdala, which have an important role in insulin resistance in this brain region. Overexpressed PKCθ in the CeA (central nucleus of amygdala) of rats increases weight gain, food intake, insulin resistance and hepatic triglycerides content. The inhibition of ER stress ameliorates insulin action/signaling, increases oxytocin and decreases NPY gene expression in the amygdala of high fat feeding rodents. Those data suggest that PKCθ and ER stress are main mechanisms of insulin resistance in the amygdala of obese rats and play an important role regulating feeding behavior.

Two Novel Mutations In The Thyroid Hormone Receptor β In Patients With Resistance To Thyroid Hormone (rth β): Clinical, Biochemical, And Molecular Data.

The syndrome of resistance to thyroid hormone (RTH β) is an inherited disorder characterized by variable tissue hyposensitivity to 3,5,30-l-triiodothyronine (T3), with persistent elevation of free-circulating T3 (FT3) and free thyroxine (FT4) levels in association with nonsuppressed serum thyrotropin (TSH). Clinical presentation is variable and the molecular analysis of THRB gene provides a short cut diagnosis. Here, we describe 2 cases in which RTH β was suspected on the basis of laboratory findings. The diagnosis was confirmed by direct THRB sequencing that revealed 2 novel mutations: the heterozygous p.Ala317Ser in subject 1 and the heterozygous p.Arg438Pro in subject 2. Both mutations were shown to be deleterious by SIFT, PolyPhen, and Align GV-GD predictive methods.

About The Article: Effect Of Combined Aerobic And Resistance Training Versus Aerobic Training On Arterial Stiffness.

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High Prevalence Of Insulin Resistance Assessed By The Glucose Clamp Technique In Hormonal And Non-hormonal Contraceptive Users.

Objective To assess the prevalence of insulin resistance (IR) and associated factors in contraceptive users. Methods A total of 47 women 18 to 40 years of age with a body mass index (kg/m(2)) < 30, fasting glucose levels < 100 mg/dl and 2-hour glucose level < 140 mg/dl after a 75-g oral glucose load were submitted to a hyperinsulinemic-euglycemic clamp. The women were distributed in tertiles regarding M-values. The analysed variables were use of combined hormonal/non-hormonal contraception, duration of use, body composition, lipid profile, glucose levels and blood pressure. Results IR was detected in 19% of the participants. The women with low M-values presented significantly higher body fat mass, waist-to-hip ratio, fasting insulin, HOMA-IR and were nulligravida, showed > 1 year of contraceptive use and higher triglyceride levels. IR was more frequent among combined oral contraceptive users, however no association was observed after regression analysis. Conclusions The prevalence of IR was high among healthy women attending a family planning clinic independent of the contraceptive method used with possible long-term negative consequences regarding their metabolic and cardiovascular health. Although an association between hormonal contraception and IR could not be found this needs further research. Family planning professionals should be proactive counselling healthy women about the importance of healthy habits.