Modification of the Tet-On regulatory system prevents the conditional-live HIV-1 variant from losing doxycycline-control

BACKGROUND: We have previously constructed a doxycycline (dox)-dependent HIV-1 variant by incorporating the Tet-On gene regulatory system into the viral genome. Replication of this HIV-rtTA virus is driven by the dox-inducible transactivator protein rtTA, and can be switched on and off at will. We proposed this conditional-live virus as a novel vaccine approach against HIV-1. Upon vaccination, replication of HIV-rtTA can be temporarily activated by transient dox administration and controlled to the extent needed for optimal induction of the immune system. However, subsequent dox-withdrawal may impose a selection for virus variants with reduced dox-dependence. RESULTS: We simulated this on/off switching of virus replication in multiple, independent cultures and could indeed select for HIV-rtTA variants that replicated without dox. Nearly all evolved variants had acquired a typical amino acid substitution at position 56 in the rtTA protein. We developed a novel rtTA variant that blocks this undesired evolutionary route and thus prevents HIV-rtTA from losing dox-control. CONCLUSION: The loss of dox-control observed upon evolution of the dox-dependent HIV-1 variant was effectively blocked by modification of the Tet-On regulatory system

HIV-1 can escape from RNA interference by evolving an alternative structure in its RNA genome

HIV-1 replication can be efficiently inhibited by intracellular expression of an siRNA targeting the viral RNA. However, HIV-1 escape variants emerged after prolonged culturing. These RNAi-resistant viruses contain nucleotide substitutions or deletions in or near the targeted sequence. We observed an inverse correlation between the level of resistance and the stability of the siRNA/target-RNA duplex. However, two escape variants showed a higher level of resistance than expected based on the duplex stability. We demonstrate that these mutations induce alternative folding of the RNA such that the target sequence is occluded from binding to the siRNA, resulting in reduced RNAi efficiency. HIV-1 can thus escape from RNAi-mediated inhibition not only through nucleotide substitutions or deletions in the siRNA target sequence, but also through mutations that alter the local RNA secondary structure. The results highlight the enormous genetic flexibility of HIV-1 and provide detailed molecular insight into the sequence specificity of RNAi and the impact of target RNA secondary structure

A Study of the PDGF Signaling Pathway with PRISM

In this paper, we apply the probabilistic model checker PRISM to the analysis of a biological system -- the Platelet-Derived Growth Factor (PDGF) signaling pathway, demonstrating in detail how this pathway can be analyzed in PRISM. We show that quantitative verification can yield a better understanding of the PDGF signaling pathway.Comment: In Proceedings CompMod 2011, arXiv:1109.104

Oncologic outcomes of screen-detected and non-screen-detected T1 colorectal cancers

Background:The incidence of T1 colorectal cancer (CRC) has increased with the implementation of CRC screening programs. It is unknown whether the outcomes and risk models for T1 CRC based on non-screen-detected patients can be extrapolated to screen-detected T1 CRC. This study aimed to compare the stage distribution and oncologic outcomes of T1 CRC patients within and outside the screening program. Methods: Data from T1 CRC patients diagnosed between 2014 and 2017 were collected from 12 hospitals in the Netherlands. The presence of lymph node metastasis (LNM) at diagnosis was compared between screen-detected and non-screen-detected patients using multivariable logistic regression. Cox proportional hazard regression was used to analyze differences in the time to recurrence (TTR), metastasis-free survival (MFS), cancer-specific survival (CSS), and overall survival. Additionally, the performance of conventional risk factors for LNM was evaluated across the groups. Results: 1803 patients were included (1114 [62%] screendetected), with median follow-up of 51 months (interquartile range 30). The proportion of LNM did not significantly differ between screen- and non-screen-detected patients (12.6% vs. 8.9%; odds ratio 1.41; 95%CI 0.89-2.23); a prediction model for LNM performed equally in both groups. The 3- and 5-year TTR, MFS, and CSS were similar for patients within and outside the screening program. However, overall survival was significantly longer in screen-detected T1 CRC patients (adjusted hazard ratio 0.51; 95%CI 0.38- 0.68). Conclusions: Screen-detected and non-screen-detected T1 CRCs have similar stage distributions and oncologic outcomes and can therefore be treated equally. However, screen-detected T1 CRC patients exhibit a lower rate of non-CRC-related mortality, resulting in longer overall survival.</p

A trial of a job-specific workers' health surveillance program for construction workers: study protocol

<p>Abstract</p> <p>Background</p> <p>Dutch construction workers are offered periodic health examinations. This care can be improved by tailoring this workers health surveillance (WHS) to the demands of the job and adjust the preventive actions to the specific health risks of a worker in a particular job. To improve the quality of the WHS for construction workers and stimulate relevant job-specific preventive actions by the occupational physician, we have developed a job-specific WHS. The job-specific WHS consists of modules assessing both physical and psychological requirements. The selected measurement instruments chosen, are based on their appropriateness to measure the workers' capacity and health requirements. They include a questionnaire and biometrical tests, and physical performance tests that measure physical functional capabilities. Furthermore, our job-specific WHS provides occupational physicians with a protocol to increase the worker-behavioural effectiveness of their counselling and to stimulate job-specific preventive actions. The objective of this paper is to describe and clarify our study to evaluate the behavioural effects of this job-specific WHS on workers and occupational physicians.</p> <p>Methods/Design</p> <p>The ongoing study of bricklayers and supervisors is a nonrandomised trial to compare the outcome of an intervention (job-specific WHS) group (n = 206) with that of a control (WHS) group (n = 206). The study includes a three-month follow-up. The primary outcome measure is the proportion of participants who have undertaken one or more of the preventive actions advised by their occupational physician in the three months after attending the WHS. A process evaluation will be carried out to determine context, reach, dose delivered, dose received, fidelity, and satisfaction. The present study is in accordance with the TREND Statement.</p> <p>Discussion</p> <p>This study will allow an evaluation of the behaviour of both the workers and occupational physician regarding the preventive actions undertaken by them within the scope of a job-specific WHS.</p> <p>Trial registration</p> <p><a href="http://www.trialregister.nl/trialreg/admin/rctview.asp?TC=3012">NTR3012</a></p

Incidence of Interval Colorectal Cancer After Negative Results From First-Round Fecal Immunochemical Screening Tests, by Cutoff Value and Participant Sex and Age

Background & Aims: We evaluated the incidence of interval cancers between the first and second rounds of colorectal cancer (CRC) screening with the FOB-Gold fecal immunochemical test (FIT), and the effects of different cutoff values and patient sex and age. Methods: We collected data from participants in a population-based

Birth weight in a large series of triplets

Background: Triplets are often born premature and with a low birth weight. Because the incidence of triplet births is rare, there are relatively few studies describing triplet birth weight characteristics. Earlier studies are often characterized by small sample sizes and lack information on important background variables such as zygosity. The objective of this study is to examine factors associated with birth weight in a large, population-based sample of triplets registered with the Netherlands Twin Register (NTR).Methods: In a sample of 1230 triplets from 410 families, the effects of assisted reproductive techniques, zygosity, birth order, gestational age, sex, maternal smoking and alcohol consumption during pregnancy on birth weight were assessed. The resemblance among triplets for birth weight was estimated as a function of zygosity. Birth weight discordance within families was studied by the pair-wise difference between triplets, expressed as a percentage of the birth weight of the heaviest child. We compare data from triplets registered with the NTR with data from population records, which include live births, stillbirths and children that have deceased within days after birth.Results: There was no effect of assisted reproductive techniques on triplet birth weight. At gestational age 24 to 40 weeks triplets gained on average 130 grams per week; boys weighed 110 grams more than girls and triplets of smoking mothers weighted 104 grams less than children of non-smoking mothers. Monozygotic triplets had lower birth weights than di- and trizygotic triplets and birth weight discordance was smaller in monozygotic triplets than in di- and trizygotic triplets. The correlation in birth weight among monozygotic and dizygotic triplets was 0.42 and 0.32, respectively. In nearly two-thirds of the families, the heaviest and the lightest triplet had a birth weight discordance over 15%. The NTR sample is representative for the Dutch triplet population that is still alive 28 days after birth.Conclusion: Birth weight is an important determinant of childhood development. Triplet status, gestational age, sex, zygosity and maternal smoking affect birth weight. The combined effects amount to a difference of 364 grams between monozygotic girl triplets of smoking mothers compared to dizygotic boy triplets of non-smoking mothers of the same gestational age. Birth weight in triplets is also influenced by genetic factors, as indicated by a larger correlation in monozygotic than in di- and trizygotic triplets

Alternative tRNA Priming of Human Immunodeficiency Virus Type 1 Reverse Transcription Explains Sequence Variation in the Primer-Binding Site That Has Been Attributed to APOBEC3G Activity

It is generally assumed that human immunodeficiency virus type 1 (HIV-1) uses exclusively the cellular [Formula: see text] molecule as a primer for reverse transcription. We demonstrate that HIV-1 uses not only [Formula: see text] but also an alternative tRNA primer. This tRNA was termed [Formula: see text] , and the near completion of the human genome project has allowed the identification of four [Formula: see text] encoding genes. Priming with [Formula: see text] results in a single nucleotide polymorphism in the viral primer-binding site that is present in multiple natural and laboratory HIV isolates. This sequence variation was recently attributed to APOBEC3G activity. However, our results show that alternative tRNA priming can cause this mutation in the absence of APOBEC3G

Improving the Safety of a Conditional-Live Human Immunodeficiency Virus Type 1 Vaccine by Controlling both Gene Expression and Cell Entry

Live attenuated human immunodeficiency virus type 1 (HIV-1) vaccines are considered unsafe because faster-replicating pathogenic virus variants may evolve after vaccination. We previously presented a conditional-live HIV-1 variant of which replication can be switched off as an alternative vaccination strategy. To improve the safety of such a vaccine, we constructed a new HIV-1 variant that depends not only on doxycycline for gene expression but also on the T20 peptide for cell entry. Replication of this virus can be limited to the level required to induce the immune system by transient administration of doxycycline and T20. Subsequent withdrawal of these inducers efficiently blocks viral replication and evolution

It is generally assumed that human immunodeficiency virus type 1 (HIV-1) uses exclusively the cellular

tRNA3 molecule as a primer for reverse transcription. We demonstrate that HIV-1 uses not only tRNA3 but Lys also an alternative tRNA primer. This tRNA was termed tRNA5, and the near completion of the huma