Antiretroviral resistance mutations in human immunodeficiency virus type 1 infected patients enrolled in genotype testing at the Central Public Health Laboratory, São Paulo, Brazil: preliminary results

Antiretroviral resistance mutations (ARM) are one of the major obstacles for pharmacological human immunodeficiency virus (HIV) suppression. Plasma HIV-1 RNA from 306 patients on antiretroviral therapy with virological failure was analyzed, most of them (60%) exposed to three or more regimens, and 28% of them have started therapy before 1997. The most common regimens in use at the time of genotype testing were AZT/3TC/nelfinavir, 3TC/D4T/nelfinavir and AZT/3TC/efavirenz. The majority of ARM occurred at protease (PR) gene at residue L90 (41%) and V82 (25%); at reverse transcriptase (RT) gene, mutations at residue M184 (V/I) were observed in 64%. One or more thymidine analogue mutations were detected in 73%. The number of ARM at PR gene increased from a mean of four mutations per patient who showed virological failure at the first ARV regimens to six mutations per patient exposed to six or more regimens; similar trend in RT was also observed. No differences in ARM at principal codon to the three drug classes for HIV-1 clades B or F were observed, but some polymorphisms in secondary codons showed significant differences. Strategies to improve the cost effectiveness of drug therapy and to optimize the sequencing and the rescue therapy are the major health priorities.

Fatigue resistance of the bond of a glass-infiltrated alumina ceramic to human dentin

Purpose: To evaluate the fatigue resistance of the bond between dentin and glass-infiltrated alumina ceramic, using different luting protocols. Materials and Methods: The null hypothesis is that the fatigue resistance varies with the luting strategy. Forty blocks of In-Ceram Alumina were prepared, and one surface of each block was abraded with 110-μm aluminum oxide particles. Then, the blocks were luted to flat dentin surfaces of 40 human third molars, using 4 different luting strategies (luting system [LS]/ceramic surface conditioning [CSC]) (n=10): (G1) [LS] RelyX-Unicem/[CSC] airborne abrasion with 110-μm Al2O3 particles; (G2) [LS] One-Step + Duo-Link (bis-GMA-based resin)/[CSC] etching with 4% hydrofluoric acid + silane agent; (G3) [LS] ED-Primer + Panavia F (MDP-based resin)/[CSC] Al2O 3; (G4) [LS] Scotchbond1+RelyX-ARC (bis-GMA-based resin)/[CSC] chairside tribochemical silica coating (air abrasion with 30-μm SiO x particles + silane). After 24 h of water storage at 37°C, the specimens were subjected to 106 fatigue cycles in shear with a sinusoidal load (0 to 21 N, 8 Hz frequency, 37°C water). A fatigue survivor score was given, considering the number of the fatigue cycles until fracture. The failure modes of failed specimens were observed in a SEM. Results: G3 (score = 5.9, 1 failure) and G4 (score = 6, no failures) were statistically similar (p = 0.33) and had significantly higher fatigue resistance than G1 (score = 3.9, 5 failures) and G2 (score = 3.7, 6 failures) (p < 0.03). SEM analysis of fractured specimens of G1 and G2 showed that almost all the failures were between ceramic and cement. Conclusion: The MDP-based resin cement + sandblasting with Al2O3 particles (G3) and bis-GMA-based resin cement + tribochemical silica coating (G4), both using the respective dentin bonding systems, were the best luting protocols for the alumina ceramic. The null hypothesis was confirmed.

Impact of insecticide resistance on the field control of Aedes aegypti in the State of São Paulo

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Genetic determinants of methicillin resistance and virulence among Staphylococcus aureus isolates recovered from clinical and surveillance cultures in a Brazilian teaching hospital

Aims. To quantify the presence of SCCmec types and virulence genes among Staphylococcus aureus colonizing and infecting patients from a teaching hospital. Methods. We analyzed 225 and 84 S. aureus isolates recovered from surveillance and clinical cultures, respectively. Strains were studied for the presence and type of SCCmec, as well as for several virulence genes. Univariate and multivariable analysis were performed in order to identify predictors of invasiveness (defined as isolation from clinical cultures). Results. The presence of SCCmec types III (OR, 2.19, 95% CI, 1.08-4.45) and IV (OR, 5.28 95% CI, 1.35-20.63) and of genes coding for exfoliative toxin B (etb, OR, 6.38, 95% CI, 1.48-27.46) and Panton-Valentine leukocidin (pvl, OR, 2.38, 95% CI, 1.16-4.86) was independently associated with invasiveness. Conclusions. SCCmec types III and IV and virulence genes are associated with greater invasiveness of S. aureus. Patients colonized with methicillin-resistant S. aureus, as well as with strains harboring etb or pvl, may be prone to develop invasive disease. Infection-preventing strategies should be more intensively applied to this group.

Strategies to minimize heat stress in broilers chickens

The objective of this study was to evaluate the application of early age thermal conditioning (TC) and dietary electrolyte balances (DEB) to minimize the effect of heat stress. A total of 240 Cobb 500 1-d-old male broiler chicks was randomly allocated to 24 floor pens with six replicates per treatment in a 2x2 factorial arrangement (with and without TC and with and without DEB). Dietary treatments consisted: (T1) a traditional diet without TC; (T2) traditional diet with TC; (T3) with the application of dietary electrolyte and without TC and (T4) application of dietary electrolyte with TC. The thermal conditioning was conducted at 5 d of age (36°C for 24 h), only half of the batch (120 birds). After this period, all birds were transferred to boxes of 1.5 x 3m (10 birds / box), with wood shavings reused as litter. Chicks were exposed to chronic heat stress (32°C) for 6 h from 35 to 39 d of age, in all treatments, being electronically monitored the temperature and humidity of the microclimate of the birds. Feed and water were provided ad libitum, even during periods of stress. Were measured performance data (weight gain, feed intake and feed conversion) and mortality rate. Data were subjected to ANOVA using the GLM procedures of SAS. The results of this study demonstrated no interaction effects of all evaluated parameters (performance and mortality). Therefore, no synergism occurred when both strategies (TC and DEB) were applied. Only the first week there was prejudice on broiler performance with the application of the TC and apparently the technique of early age thermal conditioning no improve the resistance of broilers to chronic heat stress. On the other hand, DEB had significantly favorable effects (P<0.05) on performance and minimized mortality immediately after application of the TC.

Exploration of the parameter space in an agent-based model of tuberculosis spread: emergence of drug resistance in developing vs developed countries

In this work we present an agent-based model for the spread of tuberculosis where the individuals can be infected with either drug-susceptible or drug-resistant strains and can also receive a treatment. The dynamics of the model and the role of each one of the parameters are explained. The whole set of parameters is explored to check their importance in the numerical simulation results. The model captures the beneficial impact of the adequate treatment on the prevalence of tuberculosis. Nevertheless, depending on the treatment parameters range, it also captures the emergence of drug resistance. Drug resistance emergence is particularly likely to occur for parameter values corresponding to less efficacious treatment, as usually found in developing countries.

Genetic Control of Mosquitoes: population suppression strategies

Over the last two decades, morbidity and mortality from malaria and dengue fever among other pathogens are an increasing Public Health problem. The increase in the geographic distribution of vectors is accompanied by the emergence of viruses and diseases in new areas. There are insufficient specific therapeutic drugs available and there are no reliable vaccines for malaria or dengue, although some progress has been achieved, there is still a long way between its development and actual field use. Most mosquito control measures have failed to achieve their goals, mostly because of the mosquito's great reproductive capacity and genomic flexibility. Chemical control is increasingly restricted due to potential human toxicity, mortality in no target organisms, insecticide resistance, and other environmental impacts. Other strategies for mosquito control are desperately needed. The Sterile Insect Technique (SIT) is a species-specific and environmentally benign method for insect population suppression, it is based on mass rearing, radiation mediated sterilization, and release of a large number of male insects. Releasing of Insects carrying a dominant lethal gene (RIDL) offers a solution to many of the drawbacks of traditional SIT that have limited its application in mosquitoes while maintaining its environmentally friendly and species-specific utility. The self-limiting nature of sterile mosquitoes tends to make the issues related to field use of these somewhat less challenging than for self-spreading systems characteristic of population replacement strategies. They also are closer to field use, so might be appropriate to consider first. The prospect of genetic control methods against mosquito vectored human diseases is rapidly becoming a reality, many decisions will need to be made on a national, regional and international level regarding the biosafety, social, cultural and ethical aspects of the use and deployment of these vector control methods.

Tradeoffs associated with constitutive and induced plant resistance against herbivory

Several prominent hypotheses have been posed to explain the immense variability among plant species in defense against herbivores. A major concept in the evolutionary ecology of plant defenses is that tradeoffs of defense strategies are likely to generate and maintain species diversity. In particular, tradeoffs between constitutive and induced resistance and tradeoffs relating these strategies to growth and competitive ability have been predicted. We performed three independent experiments on 58 plant species from 15 different plant families to address these hypotheses in a phylogenetic framework. Because evolutionary tradeoffs may be altered by human-imposed artificial selection, we used 18 wild plant species and 40 cultivated garden-plant species. Across all 58 plant species, we demonstrate a tradeoff between constitutive and induced resistance, which was robust to accounting for phylogenetic history of the species. Moreover, the tradeoff was driven by wild species and was not evident for cultivated species. In addition, we demonstrate that more competitive species—but not fast growing ones—had lower constitutive but higher induced resistance. Thus, our multispecies experiments indicate that the competition–defense tradeoff holds for constitutive resistance and is complemented by a positive relationship of competitive ability with induced resistance. We conclude that the studied genetically determined tradeoffs are indeed likely to play an important role in shaping the high diversity observed among plant species in resistance against herbivores and in life history traits.

Teacher resistance to school-based consultation with the school psychologist: An analysis according to school level and model of identification for special education services

Consultation is promoted throughout school psychology literature as a best practice in service delivery. This method has numerous benefits including being able to work with more students at one time, providing practitioners with preventative rather than strictly reactive strategies, and helping school professionals meet state and federal education mandates and initiatives. Despite the benefits of consultation, teachers are sometimes resistant to this process.This research studies variables hypothesized to lead to resistance (Gonzalez, Nelson, Gutkin, & Shwery, 2004) and attempts to distinguish differences between school level (elementary, middle and high school) with respect to the role played by these variables and to determine if the model used to identify students for special education services has an influence on resistance factors. Twenty-sixteachers in elementary and middle schools responded to a demographicquestionnaire and a survey developed by Gonzalez, et al. (2004). This survey measures eight variables related to resistance to consultation. No high school teachers responded to the request to participate. Results of analysis of variance indicated a significant difference in the teaching efficacy subscale with elementary teachers reporting more efficacy in teaching than middle school teachers. Results also indicate a significant difference in classroom managementefficacy with teachers who work in schools that identify students according to a Response to Intervention model reporting higher classroom management efficacy than teachers who work in schools that identify students according to a combined method of refer-test-place/RtI combination model. Implications, limitations and directions for future research are discussed.

Inhibition of the AKT/mTOR and erbB pathways by gefitinib, perifosine and analogs of gonadotropin-releasing hormone I and II to overcome tamoxifen resistance in breast cancer cells

Endocrine resistance in breast cancer remains a major clinical problem and is caused by crosstalk mechanisms of growth factor receptor cascades, such as the erbB and PI3K/AKT pathways. The possibilities a single breast cancer cell has to achieve resistance are manifold. We developed a model of 4-hydroxy-tamoxifen (OHT)‑resistant human breast cancer cell lines and compared their different expression patterns, activation of growth factor receptor pathways and compared cells by genomic hybridization (CGH). We also tested a panel of selective inhibitors of the erbB and AKT/mTOR pathways to overcome OHT resistance. OHT‑resistant MCF-7-TR and T47D-TR cells showed increased expression of HER2 and activation of AKT. T47D-TR cells showed EGFR expression and activated MAPK (ERK-1/2), whereas in resistant MCF-7-TR cells activated AKT was due to loss of CTMP expression. CGH analyses revealed remarkable aberrations in resistant sublines, which were predominantly depletions. Gefitinib inhibited erbB signalling and restored OHT sensitivity in T47D-TR cells. The AKT inhibitor perifosine restored OHT sensitivity in MCF-7-TR cells. All cell lines showed expression of receptors for gonadotropin-releasing hormone (GnRH) I and II, and analogs of GnRH-I/II restored OHT sensitivity in both resistant cell lines by inhibition of erbB and AKT signalling. In conclusion, mechanisms to escape endocrine treatment in breast cancer share similarities in expression profiling but are based on substantially different genetic aberrations. Evaluation of activated mediators of growth factor receptor cascades is helpful to predict response to specific inhibitors. Expression of GnRH-I/II receptors provides multi-targeting treatment strategies.

Protective autophagy is involved in resistance towards MET inhibitors in human gastric adenocarcinoma cells

MET, also known as hepatocyte growth factor receptor (HGFR), is a receptor tyrosine kinase with an important role, both in normal cellular function as well as in oncogenesis. In many cancer types, abnormal activation of MET is related to poor prognosis and various strategies to inhibit its function, including small molecule inhibitors, are currently in preclinical and clinical evaluation. Autophagy, a self-digesting recycling mechanism with cytoprotective functions, is induced by cellular stress. This process is also induced upon cytotoxic drug treatment of cancer cells and partially allows these cells to escape cell death. Thus, since autophagy protects different tumor cells from chemotherapy-induced cell death, current clinical trials aim at combining autophagy inhibitors with different cancer treatments. We found that in a gastric adenocarcinoma cell line GTL-16, where MET activity is deregulated due to receptor overexpression, two different MET inhibitors PHA665752 and EMD1214063 lead to cell death paralleled by the induction of autophagy. A combined treatment of MET inhibitors together with the autophagy inhibitor 3-MA or genetically impairing autophagy by knocking down the key autophagy gene ATG7 further decreased cell viability of gastric cancer cells. In general, we observed the induction of cytoprotective autophagy in MET expressing cells upon MET inhibition and a combination of MET and autophagy inhibition resulted in significantly decreased cell viability in gastric cancer cells.

Equine parasite control under prescription-only conditions in Denmark - Awareness, knowledge, perception, and strategies applied.

Due to widespread development of anthelmintic resistance in equine parasites, recommendations for their control are currently undergoing marked changes with a shift of emphasis toward more coprological surveillance and reduced treatment intensity. Denmark was the first nation to introduce prescription-only restrictions of anthelmintic drugs in 1999, but other European countries have implemented similar legislations over recent years. A questionnaire survey was performed in 2008 among Danish horse owners to provide a current status of practices and perceptions with relation to parasite control. Questions aimed at describing the current use of coprological surveillance and resulting anthelmintic treatment intensities, evaluating knowledge and perceptions about the importance of various attributes of parasite control, and assessing respondents' willingness to pay for advice and parasite surveillance services from their veterinarians. A total of 1060 respondents completed the questionnaire. A large majority of respondents (71.9%) were familiar with the concept of selective therapy. Results illustrated that the respondents' self-evaluation of their knowledge about parasites and their control associated significantly with their level of interest in the topic and their type of education (P<0.0001). The large majority of respondents either dewormed their horses twice a year and/or performed two fecal egg counts per horse per year. This approach was almost equally pronounced in foals, horses aged 1-3 years old, and adult horses. The respondents rated prevention of parasitic disease and prevention of drug resistance as the most important attributes, while cost and frequent fecal testing were rated least important. Respondents' actual spending on parasite control per horse in the previous year correlated significantly with the amount they declared themselves willing to spend (P<0.0001). However, 44.4% declared themselves willing to pay more than what they were spending. Altogether, results indicate that respondents were generally familiar with equine parasites and the concept of selective therapy, although there was some confusion over the terms small and large strongyles. They used a large degree of fecal surveillance in all age groups, with a majority of respondents sampling and/or treating around twice a year. Finally, respondents appeared willing to spend money on parasite control for their horses. It is of concern that the survey suggested that foals and young horses are treated in a manner very similar to adult horses, which is against current recommendations. Thus, the survey illustrates the importance of clear communication of guidelines for equine parasite control.

Strategies to increase the use and approval of gender-fair language

When masculine forms are used to refer to men and women, this causes male-biased cognitive representations and behavioral consequences, as numerous studies have shown. This effect can be avoided or reduced with the help of gender-fair language. In this talk, we will present different approaches that aim at influencing people’s use of and attitudes towards gender-fair language. Firstly, we tested the influence of gender-fair input on people’s own use of gender-fair language. Based on Irmen and Linner’s (2005) adaptation of the scenario mapping and focus approach (Sanford & Garrod, 1998), we found that after reading a text with gender-fair forms women produced more gender-fair forms than women who read gender-neutral texts or texts containing masculine generics. Men were not affected. Secondly, we examined reactions to arguments which followed the Elaboration Likelihood Model (Petty &Cacioppo, 1986). We assumed that strong pros and cons would be more effective than weak arguments or control statements. The results indicated that strong pros could convince some, but not all participants, suggesting a complex interplay of diverse factors in reaction to attempts at persuasion. The influence of people’s initial characteristics will be discussed. Currently, we are investigating how self-generated refutations, in addition to arguments, may influence initial attitudes. Based on the resistance appraisal hypothesis (Tormala, 2008), we assume that individuals are encouraged in their initial attitude if they manage to refute strong counter-arguments. The results of our studies will be discussed regarding their practical implications.

MECHANISM-BASED STRATEGIES TO ENHANCE THE ACTIONS OF A

Heat shock protein 90 (HSP90) is an abundant molecular chaperone that regulates the functional stability of client oncoproteins, such as STAT3, Raf-1 and Akt, which play a role in the survival of malignant cells. The chaperone function of HSP90 is driven by the binding and hydrolysis of ATP. The geldanamycin analog, 17-AAG, binds to the ATP pocket of HSP90 leading to the degradation of client proteins. However, treatment with 17-AAG results in the elevation of the levels of antiapoptotic proteins HSP70 and HSP27, which may lead to cell death resistance. The increase in HSP70 and HSP27 protein levels is due to the activation of the transcription factor HSF-1 binding to the promoter region of HSP70 and HSP27 genes. HSF-1 binding subsequently promotes HSP70 and HSP27 gene expression. Based on this, I hypothesized that inhibition of transcription/translation of HSP or client proteins would enhance 17-AAG-mediated cytotoxicity. Multiple myeloma (MM) cell lines MM.1S, RPMI-8226, and U266 were used as a model. To test this hypothesis, two different strategies were used. For the first approach, a transcription inhibitor was combined with 17-AAG. The established transcription inhibitor Actinomycin D (Act D), used in the clinic, intercalates into DNA and blocks RNA elongation. Stress inducible (HSP90á, HSP70 and HSP27) and constitutive (HSP90â and HSC70) mRNA and protein levels were measured using real time RT-PCR and immunoblot assays. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the MM cell lines. This induction of HSP mRNA levels was diminished by 0.05 µg/mL Act D for 12 hours in the combination treatment, except for HSP70. At the protein level, Act D abrogated the 17-AAG-mediated induction of all HSP expression levels, including HSP70. Cytotoxic evaluation (Annexin V/7-AAD assay) of Act D in combination with 17-AAG suggested additive or more than additive interactions. For the second strategy, an agent that affected bioenergy production in addition to targeting transcription and translation was used. Since ATP is necessary for the proper folding and maturation of client proteins by HSP90, ATP depletion should lead to a decrease in client protein levels. The transcription and translation inhibitor 8-Chloro-Adenosine (8-Cl-Ado), currently in clinical trials, is metabolized into its cytotoxic form 8-Cl-ATP causing a parallel decrease of the cellular ATP pool. Treatment with 0.5 µM 17-AAG for 8 hours resulted in the induction of all HSP transcript and protein levels in the three MM cell lines evaluated. In the combination treatment, 10 µM 8-Cl-Ado for 20 hours did not abrogate the induction of HSP mRNA or protein levels. Since cellular bioenergy is necessary for the stabilization of oncoproteins by HSP90, immunoblot assays analyzing for expression levels of client proteins such as STAT3, Raf-1, and Akt were performed. Immunoblot assays detecting for the phosphorylation status of the translation repressor 4E-BP1, whose activity is modulated by upstream kinases sensitive to changes in ATP levels, were also performed. The hypophosphorylated state of 4E-BP1 leads to translation repression. Data indicated that treatment with 17-AAG alone resulted in a minor (<10%) change in STAT3, Raf-1, and Akt protein levels, while no change was observed for 4E-BP1. The combination treatment resulted in more than 50% decrease of the client protein levels and hypophosphorylation of 4E-BP1 in all MM cell lines. Treatment with 8-Cl-Ado alone resulted in less than 30% decrease in client protein levels as well as a decrease in 4E-BP1 phosphorylation. Cytotoxic evaluation of 8-Cl-Ado in combination with 17-AAG resulted in more than additive cytotoxicity when drugs were combined in a sequential manner. In summary, these data suggest that the mechanism-based combination of agents that target transcription, translation, or decrease cellular bioenergy with 17-AAG results in increase cytotoxicity when compared to the single agents. Such combination strategies may be applied in the clinic since these drugs are established chemotherapeutic agents or currently in clinical trials.

Strategies for reducing fumigant loss to the atmosphere

A model is developed to describe transport and loss of methyl bromide (MeBr) in soil following application as a soil fumigant. The model is used to investigate the effect of soil and management factors on MeBr volatilization. Factors studied include depth of injection, soil water content, presence or absence of tarp, depth to downward barrier, and irrigation after injection. Of these factors, the most important was irrigation after injection followed by covering with the tarp, which increased the diffusive resistance of the soil and prevented early loss of MeBr. The model offers an explanation for the apparently contradictory observations of earlier field studies of MeBr volatilization from soils under different conditions. The model was also used to calculate the concentration-time index for various management alternatives, showing that the irrigation application did not make the surface soil more difficult to fumigate, except at very early times. Therefore, irrigation shows promise for reducing fumigant loss while at the same time permitting control of target organisms during fumigation.