The first strand transfer reaction of HIV-1 reverse transcription is more efficient in infected cells than in cell-free natural endogenous reverse transcription reactions

Background: In the presence of dNTPs, intact HIV-1 virions are capable of reverse transcribing at least part of their genome, a process known as natural endogenous reverse transcription (NERT). PCR analysis of virion DNA produced by NERT revealed that the first strand transfer reaction (1stST) was inefficient in intact virions, with minus strand (-) strong stop DNA (ssDNA) copy numbers up to 200 times higher than post-1stST products measured using primers in U3 and U5. This was in marked contrast to the efficiency of 1stST observed in single-round cell infection assays, in which (-) ssDNA and U3-U5 copy numbers were indistinguishable. Objectives: To investigate the reasons for the discrepancy in first strand transfer efficiency between intact cell-free virus and the infection process. Study design: Alterations of both NERT reactions and the conditions of cell infection were used to test whether uncoating and/or entry play a role in the discrepancy in first strand transfer efficiency. Results and Conclusions: The difference in 1stST efficiency could not be attributed simply to viral uncoating, since addition of very low concentrations of detergent to NERT reactions removed the viral envelope without disrupting the reverse transcription complex, and these conditions resulted in no improvement in 1stST efficiency. Virus pseudotyped with surface glycoproteins from either vesicular stomatitis virus or amphotrophic murine leukaemia virus also showed low levels of 1stST in low detergent NERT assays and equivalent levels of (-) ssDNA and 1stST in single-round infections of cells, demonstrating that the gp120-mediated infection process did not select for virions capable of carrying out 1stST. These data indicate that a post-entry event or factor may be involved in efficient HIV-1 reverse transcription in vivo. (C) 2002 Elsevier Science B.V. All rights reserved.

GUIsurfer: A Reverse Engineering Framework for User Interface Software

GUIsurfer: A Reverse Engineering Framework for User Interface Software

The GUISurfer tool: towards a language independent approach to reverse engineering GUI code

Graphical user interfaces (GUIs) are critical components of today's software. Developers are dedicating a larger portion of code to implementing them. Given their increased importance, correctness of GUIs code is becoming essential. This paper describes the latest results in the development of GUISurfer, a tool to reverse engineer the GUI layer of interactive computing systems. The ultimate goal of the tool is to enable analysis of interactive system from source code.

Combining Formal Methods and Functional Strategies Regarding the Reverse Engineering of Interactive Applications

Abstract. Graphical user interfaces (GUIs) make software easy to use by providing the user with visual controls. Therefore, correctness of GUI’s code is essential to the correct execution of the overall software. Models can help in the evaluation of interactive applications by allowing designers to concentrate on its more important aspects. This paper describes our approach to reverse engineer an abstract model of a user interface directly from the GUI’s legacy code. We also present results from a case study. These results are encouraging and give evidence that the goal of reverse engineering user interfaces can be met with more work on this technique.

Models for the Reverse Engineering of Java/Swing Applications

Abstract. Interest in design and development of graphical user interface (GUIs) is growing in the last few years. However, correctness of GUI's code is essential to the correct execution of the overall software. Models can help in the evaluation of interactive applications by allowing designers to concentrate on its more important aspects. This paper describes our approach to reverse engineering abstract GUI models directly from the Java/Swing code.

Production of hydroxamic acids by immobilized Pseudomonas aeruginosa cells Kinetic analysis in reverse micelles

Intact cells from Pseudomonas aeruginosa strain L10 containing amidase were used as biocatalysts both free and immobilized in a reverse micellar system. The apparent kinetic constants for the transamidation reaction in hydroxamic acids synthesis, were determined using substrates such as aliphatic, amino acid and aromatic amides and esters, in both media. In reverse micelles, K-m values decreased 2-7 fold relatively to the free biocatalyst using as substrates acetamide, acrylamide, propionamide and glycinamide ethyl ester. We have concluded that overall the affinity of the biocatalyst to each substrate increases when reactions are performed in the reversed micellar system as opposed to the buffer system. The immobilized biocatalyst in general, exhibits higher stability and faster rates of reactions at lower substrates concentration relatively to the free form, which is advantageous. Additionally, the immobilization revealed to be suitable for obtaining the highest yields of hydroxamic acids derivatives, in some cases higher than 80%. (C) 2013 Elsevier B.V. All rights reserved.

Logistic optimization in tourism networks

55th European Regional Science Association Congress, Lisbon, Portugal (25-28 August 2015).

Kinetic behavior of Desulfovibrio gigas aldehyde oxidoreductase encapsulated in reverse micelles

Biochemical and Biophysical Research Communications 308 (2003) 73–78

Hydrogen evolution and consumption in AOT–isooctane reverse micelles by Desulfovibrio gigas hydrogenase

The enzyme hydrogenase isolated from the sulphate reducing anaerobic bacterium Desulfovibrio gigas was encapsulated in reverse micelles of AOT–water–isooctane. The enzyme ability to consume molecular hydrogen was studied as a function of the micelle size (given by Wo = [H2O]/[organic solvent]). A peak of catalytic activity was obtained for Wo = 18, a micelle size theoretically fitting the heterodimeric hydrogenase molecule. At this Wo value, the recorded catalytic activity was slightly higher than in a buffer system(Kcat = 169.43 s−1 against the buffer value of 151 s−1). The optimal buffer used to encapsulate the enzyme was found to be imidazole 50 mM, pH 9.0. The molecular hydrogen production activity was also tested in this reverse micelle medium.

Strength improvement of adhesively-bonded joints using a reverse-bent geometry

Adhesive bonding of components has become more efficient in recent years due to the developments in adhesive technology, which has resulted in higher peel and shear strengths, and also in allowable ductility up to failure. As a result, fastening and riveting methods are being progressively replaced by adhesive bonding, allowing a big step towards stronger and lighter unions. However, single-lap bonded joints still generate substantial peel and shear stress concentrations at the overlap edges that can be harmful to the structure, especially when using brittle adhesives that do not allow plasticization in these regions. In this work, a numerical and experimental study is performed to evaluate the feasibility of bending the adherends at the ends of the overlap for the strength improvement of single-lap aluminium joints bonded with a brittle and a ductile adhesive. Different combinations of joint eccentricity were tested, including absence of eccentricity, allowing the optimization of the joint. A Finite Element stress and failure analysis in ABAQUS® was also carried out to provide a better understanding of the bent configuration. Results showed a major advantage of using the proposed modification for the brittle adhesive, but the joints with the ductile adhesive were not much affected by the bending technique.

Reverse problem-based learning - a case study with a Braille machine

Engineering Education includes not only teaching theoretical fundamental concepts but also its verification during practical lessons in laboratories. The usual strategies to carry out this action are frequently based on Problem Based Learning, starting from a given state and proceeding forward to a target state. The possibility or the effectiveness of this procedure depends on previous states and if the present state was caused or resulted from earlier ones. This often happens in engineering education when the achieved results do not match the desired ones, e.g. when programming code is being developed or when the cause of the wrong behavior of an electronic circuit is being identified. It is thus important to also prepare students to proceed in the reverse way, i.e. given a start state generate the explanation or even the principles that underlie it. Later on, this sort of skills will be important. For instance, to a doctor making a patient?s story or to an engineer discovering the source of a malfunction. This learning methodology presents pedagogical advantages besides the enhanced preparation of students to their future work. The work presented on his document describes an automation project developed by a group of students in an engineering polytechnic school laboratory. The main objective was to improve the performance of a Braille machine. However, in a scenario of Reverse Problem-Based learning, students had first to discover and characterize the entire machine's function before being allowed (and being able) to propose a solution for the existing problem.

Predicting Xerostomia induced by IMRT treatments: A logistic regression approach

Radiotherapy is one of the main treatments used against cancer. Radiotherapy uses radiation to destroy cancerous cells trying, at the same time, to minimize the damages in healthy tissues. The planning of a radiotherapy treatment is patient dependent, resulting in a lengthy trial and error procedure until a treatment complying as most as possible with the medical prescription is found. Intensity Modulated Radiation Therapy (IMRT) is one technique of radiation treatment that allows the achievement of a high degree of conformity between the area to be treated and the dose absorbed by healthy tissues. Nevertheless, it is still not possible to eliminate completely the potential treatments’ side-effects. In this retrospective study we use the clinical data from patients with head-and-neck cancer treated at the Portuguese Institute of Oncology of Coimbra and explore the possibility of classifying new and untreated patients according to the probability of xerostomia 12 months after the beginning of IMRT treatments by using a logistic regression approach. The results obtained show that the classifier presents a high discriminative ability in predicting the binary response “at risk for xerostomia at 12 months”

Detection and identification of dengue virus isolates from Brazil by a simplified reverse transcription - polymerase chain reaction (RT-PCR) method

We show here a simplified RT-PCR for identification of dengue virus types 1 and 2. Five dengue virus strains, isolated from Brazilian patients, and yellow fever vaccine 17DD as a negative control, were used in this study. C6/36 cells were infected and supernatants were collected after 7 days. The RT-PCR, done in a single reaction vessel, was carried out following a 1/10 dilution of virus in distilled water or in a detergent mixture containing Nonidet P40. The 50 µl assay reaction mixture included 50 pmol of specific primers amplifying a 482 base pair sequence for dengue type 1 and 210 base pair sequence for dengue type 2. In other assays, we used dengue virus consensus primers having maximum sequence similarity to the four serotypes, amplifying a 511 base pair sequence. The reaction mixture also contained 0.1 mM of the four deoxynucleoside triphosphates, 7.5 U of reverse transcriptase, 1U of thermostable Taq DNA polymerase. The mixture was incubated for 5 minutes at 37ºC for reverse transcription followed by 30 cycles of two-step PCR amplification (92ºC for 60 seconds, 53ºC for 60 seconds) with slow temperature increment. The PCR products were subjected to 1.7% agarose gel electrophoresis and visualized by UV light after staining with ethidium bromide solution. Low virus titer around 10 3, 6 TCID50/ml was detected by RT-PCR for dengue type 1. Specific DNA amplification was observed with all the Brazilian dengue strains by using dengue virus consensus primers. As compared to other RT-PCRs, this assay is less laborious, done in a shorter time, and has reduced risk of contamination

Diagnosis of hepatitis C virus in Brazilian blood donors using a reverse transcriptase nested polymerase chain reaction: comparison with enzyme immunoassay and recombinant protein immunoblot assay

Screening blood donations for anti-HCV antibodies and alanine aminotransferase (ALT) serum levels generally prevents the transmission of hepatitis C virus (HCV) by transfusion. The aim of the present study was to evaluate the efficiency of the enzyme immunoassay (EIA) screening policy in identifying potentially infectious blood donors capable to transmit hepatitis C through blood transfusion. We have used a reverse transcriptase (RT)-nested polymerase chain reaction (PCR) to investigate the presence of HCV-RNA in blood donors. The prevalence of HCV-RNA positive individuals was compared with the recombinant immunoblot assay (RIBA-2) results in order to assess the usefulness of both tests as confirmatory assays. Both tests results were also compared with the EIA-2 OD/C ratio (optical densities of the samples divided by the cut off value). ALT results were expressed as the ALT quotient (qALT), calculated dividing the ALT value of the samples by the maximum normal value (53UI/l) for the method. Donors (n=178) were divided into five groups according to their EIA anti-HCV status and qALT: group A (EIA > or = 3, ALT<1), group B (EIA > or = 3, ALT>1), group C (1<=EIA<3, ALT<1), group D (1<=EIA<3, ALT>1) and group E (EIA<=0.7). HCV sequences were detected by RT-nested PCR, using primers for the most conserved region of viral genome. RIBA-2 was applied to the same samples. In group A (n=6), all samples were positive by RT-nested PCR and RIBA-2. Among 124 samples in group B, 120 (96.8%) were RIBA-2 positive and 4 (3.2%) were RIBA-2 indeterminate but were seropositive for antigen c22.3. In group B, 109 (87.9%) of the RIBA-2 positive samples were also RT-nested PCR positive, as well as were all RIBA-2 indeterminate samples. In group C, all samples (n=9) were RT-nested PCR negative: 4 (44.4%) were also RIBA-2 negative, 4 (44.4%) were RIBA-2 positive and 1 (11.1%) was RIBA-2 indeterminate. HCV-RNA was detected by RT-nested PCR in 3 (37.5%) out of 8 samples in group D. Only one of them was also RIBA-2 positive, all the others were RIBA-2 indeterminate. All of the group E samples (controls) were RT- nested PCR and RIBA-2 negative. Our study suggests a strong relation between anti-HCV EIA-2 ratio > or = 3 and detectable HCV-RNA by RT-nested PCR. We have also noted that blood donors with RIBA-2 indeterminate presented a high degree of detectable HCV-RNA using RT-nested PCR (75%), especially when the c22.3 band was detected.

Analysis of HIV- type 1 protease and reverse transcriptase in Brazilian children failing highly active antiretroviral therapy (HAART)

The aim of this study was to evaluate the genotypic resistance profiles of HIV-1 in children failing highly active antiretroviral therapy (HAART). Forty-one children (median age = 67 months) receiving HAART were submitted to genotypic testing when virological failure was detected. cDNA was extracted from PBMCs and amplified by nested PCR for the reverse transcriptase and protease regions of the pol gene. Drug resistance genotypes were determined from DNA sequencing. According to the genotypic analysis, 12/36 (33.3%) and 6/36 (16.6%) children showed resistance and possible resistance, respectively, to ZDV; 5/36 (14%) and 4/36 (11.1%), respectively, showed resistance and possible resistance to ddI; 4/36 (11.1%) showed resistance to 3TC and D4T; and 3/36 (8.3%) showed resistance to Abacavir. A high percentage (54%) of children exhibited mutations conferring resistance to NNRTI class drugs. Respective rates of resistance and possible resistance to PIs were: RTV (12.2%, 7.3%); APV (2.4%, 12.1%); SQV(0%, 12.1%); IDV (14.6%, 4.9%), NFV (22%, 4.9%), LPV/RTV (2.4%, 12.1%). Overall, 37/41 (90%) children exhibited virus with mutations related to drug resistance, while 9% exhibited resistance to all three antiretroviral drug classes.