Comparison and transfer testing of multiplex ligation detection methods for GM plants

Background  

With the increasing number of GMOs on the global market the maintenance of European GMO regulations is becoming more complex. For the analysis of a single food or feed sample it is necessary to assess the sample for the presence of many GMO-targets simultaneously at a sensitive level. Several methods have been published regarding DNA-based multidetection. Multiplex ligation detection methods have been described that use the same basic approach: i) hybridisation and ligation of specific probes, ii) amplification of the ligated probes and iii) detection and identification of the amplified products. Despite they all have this same basis, the published ligation methods differ radically. The present study investigated with real-time PCR whether these different ligation methods have any influence on the performance of the probes. Sensitivity and the specificity of the padlock probes (PLPs) with the ligation protocol with the best performance were also tested and the selected method was initially validated in a laboratory exchange study.     

Heme oxygenase-1 prevents smoke induced B-cell infiltrates: a role for regulatory T cells?

Background

Smoking is the most important cause for the development of COPD. Since not all smokers develop COPD, it is obvious that other factors must be involved in disease development. We hypothesize that heme oxygenase-1 (HO-1), a protective enzyme against oxidative stress and inflammation, is insufficiently upregulated in COPD.The effects of HO-1 modulation on cigarette smoke induced inflammation and emphysema were tested in a smoking mouse model.

Methods

Mice were either exposed or sham exposed to cigarette smoke exposure for 20 weeks. Cobalt protoporphyrin or tin protoporphyrin was injected during this period to induce or inhibit HO-1 activity, respectively. Afterwards, emphysema development, levels of inflammatory cells and cytokines, and the presence of B-cell infiltrates in lung tissue were analyzed.

Results

Smoke exposure induced emphysema and increased the numbers of inflammatory cells and numbers of B-cell infiltrates, as well as the levels of inflammatory cytokines in lung tissue. HO-1 modulation had no effects on smoke induced emphysema development, or the increases in neutrophils and macrophages and inflammatory cytokines. Interestingly, HO-1 induction prevented the development of smoke induced B-cell infiltrates and increased the levels of CD4⁺CD25⁺ T cells and Foxp3 positive cells in the lungs. Additionally, the CD4⁺CD25⁺ T cells correlated positively with the number of Foxp3 positive cells in lung tissue, indicating that these cells were regulatory T cells.

Conclusion

These results support the concept that HO-1 expression influences regulatory T cells and indicates that this mechanism is involved in the suppression of smoke induced B-cell infiltrates. The translation of this interaction to human COPD should now be pursued.     

Adenovirus Transformation of Hamster Embryo Cells

Inoculation of hamster embryo cell cultures with human adenovirus type 12 (Ad12) or simian adenovirus (SA7) resulted in the formation of foci of morphologically transformed cells within 12 days. The rapid appearance of well-defined foci was dependent upon the transfer of cells into new plates, with sufficient dilution after virus adsorption, and was independent of virus dose. Dose-response studies showed linearity of focus formation with dilution of Ad12 or SA7. Results averaged from several experiments show plaque-forming unit to focus-forming unit ratios of approximately 1.8 x 10(6) for Ad12 and 2.6 x 10(5) for SA7. Other experiments showed that most of the adenovirus involved in transformation was adsorbed by 3 hr. Cell lines derived from SA7 transformed cells produced tumors within 19 days when inoculated intradermally into young adult hamsters. Such cell-induced tumors histologically resembled SA7 virus-induced hamster tumors. Formation of tumors with SA7 transformed cells was inhibited by prior immunization of test animals with SA7 or Ad12 virus.     

Structural genes of the mouse major urinary protein are on chromosome 4

The major urinary proteins (MUPs) of mouse are a family of at least three major proteins which are synthesized in the liver of all strains of mice. The relative levels of synthesis of these proteins with respect to each other in the presence of testosterone is regulated by the Mup-a locus located on chromosome 4. In an effort to determine the mechanism of this regulation in molecular terms, a cDNA clone containing most of the coding region of a MUP protein has been isolated and identified by partial DNA sequence analysis. Using a combination of hybridization analysis and somatic cell genetics, the structural gene family has been unambiguously mapped to mouse chromosome 4. These data suggest that Mup-a regulation operates in a cis fashion and that models proposing trans regulation of MUP protein synthesis are unlikely.     

Significance of treatment interval and DNA repair in the enhancement of viral transformation by chemical carcinogens and mutagens

Treatment of Syrian hamster embryo cells with diverse classes of chemical carcinogens enhanced transformation by a carcinogenic simian adenovirus, SA7. Optimal enhancement was a function of time of chemical addition in relation to time of virus addition and cell transfer. Aflatoxin B₁ (AFB₁) and the polycyclic hydrocarbons, benzo(a)pyrene (B(a)P), 3-methylcholanthrene (MCA), and 7,12-dimethylbenz(a)anthracene (DMBA) enhanced SA7 transformation when added prior to virus, but inhibited transformation when added after virus adsorption and cell transfer. The enhancement of SA7 transformation was maximal when cytosine arabinoside, caffeine and 6-acetoxy-benzo(a)pyrene (6-ac-B(a)P) were added after virus, but minimal when added before virus. A third class of chemicals, including β-propiolactone (β-PL), methyl methanesulfonate (MMS), N-acetoxy-2-acetylaminofluorene (Ac-AAF), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG), and methylazoxymethanol acetate (MAM-ac), enhanced SA7 transformation added before, or after, virus inoculation and cell transfer. All chemicals, which induced changes in DNA sedimentation in alkaline sucrose gradients and unscheduled DNA (repair) synthesis in hamster cells, increased the frequency of SA7 transformation. However, several chemicals such as dibenz(a,h)anthracene (DB(a,h)A), benzo(e)pyrene (B(e)P), cytosine arabinoside, and caffeine enhanced SA7 transformation but did not induce DNA sedimentation changes or repair. Chemicals that cause DNA damage, which can be repaired by hamster cells, may enhance viral transformation by providing additional sites for integration of viral DNA during the repair process. Chemicals that apparently do not induce DNA repair synthesis may enhance viral transformation by incorporation of viral DNA into gaps in cell DNA at sites of unrepaired damage during scheduled DNA synthesis.     

Low temperature delays timing and enhances the cost of nitrogen fixation in the unicellular cyanobacterium Cyanothece

Marine nitrogen-fixing cyanobacteria are largely confined to the tropical and subtropical ocean. It has been argued that their global biogeographical distribution reflects the physiologically feasible temperature range at which they can perform nitrogen fixation. In this study we refine this line of argumentation for the globally important group of unicellular diazotrophic cyanobacteria, and pose the following two hypotheses: (i) nitrogen fixation is limited by nitrogenase activity at low temperature and by oxygen diffusion at high temperature, which is manifested by a shift from strong to weak temperature dependence of nitrogenase activity, and (ii) high respiration rates are required to maintain very low levels of oxygen for nitrogenase, which results in enhanced respiratory cost per molecule of fixed nitrogen at low temperature. We tested these hypotheses in laboratory experiments with the unicellular cyanobacterium Cyanothece sp. {"type":"entrez-nucleotide","attrs":{"text":"BG043511","term_id":"12489990","term_text":"BG043511"}}BG043511. In line with the first hypothesis, the specific growth rate increased strongly with temperature from 18 to 30 °C, but leveled off at higher temperature under nitrogen-fixing conditions. As predicted by the second hypothesis, the respiratory cost of nitrogen fixation and also the cellular C:N ratio rose sharply at temperatures below 21 °C. In addition, we found that low temperature caused a strong delay in the onset of the nocturnal nitrogenase activity, which shortened the remaining nighttime available for nitrogen fixation. Together, these results point at a lower temperature limit for unicellular nitrogen-fixing cyanobacteria, which offers an explanation for their (sub)tropical distribution and suggests expansion of their biogeographical range by global warming.     

Health-related quality of life and functional ability in patients with early arthritis during remission steered treatment: results of the IMPROVED study

Introduction

The aim of this study was to investigate patient reported outcomes (PROs) of functional ability and health related quality of life (HRQoL) in patients with early (rheumatoid) arthritis during one year of remission steered treatment.

Methods

In this study, 610 patients with early rheumatoid arthritis (RA) or undifferentiated arthritis (UA) were treated with methotrexate (MTX) and tapered high dose of prednisone. Patients in early remission (Disease Activity Score (DAS) <1.6 after 4 months) tapered prednisone to zero and when in persistent remission, also tapered MTX. Patients not in early remission were randomized to either MTX + hydroxychloroquine + sulphasalazine + prednisone (arm 1) or to MTX + adalimumab (arm 2). Every 4 months, patients filled out the Health Assessment Questionnaire (HAQ) and the McMaster Toronto Arthritis Patient Preference Questionnaire (MACTAR), the Short Form 36 (SF-36) and visual analogue scales (VAS). Change scores were compared between treatment groups. The association with achieving remission was analyzed using linear mixed models.

Results

During year 1, patients who achieved early remission had the most improvement in PROs with scores comparable to the general population. Patients in the randomization arms showed less improvement. Scores were comparable between the arms. There was a significant association between achieving remission and scores of HAQ, MACTAR and physical HRQoL.

Conclusions

In early arthritis, PROs of functional ability and HRQoL after one year of remission steered treatment reach normal values in patients who achieved early remission. In patients not in early remission, who were randomized to two strategy arms, PROs improved less, with similar scores in both treatment arms.

Trial registrations

ISRCTN11916566 and EudraCT2006-006186-16     

(Cost)-effectiveness of a multi-component intervention for adults with epilepsy: study protocol of a Dutch randomized controlled trial (ZMILE study)

Background

In patients with epilepsy, poor adherence to anti-epileptic drugs has been shown to be the most important cause of poorly controlled epilepsy. Furthermore, it has been noted that the quality of life among patients with epilepsy can be improved by counseling and treatments aimed at increasing their self-efficacy and concordance, thus stimulating self-management skills. However, there is a need for evidence on the effectiveness of such programs, especially within epilepsy care. Therefore, we have developed a multi-component intervention (MCI) which combines a self-management/education program with e-Health interventions. Accordingly, the overall objective of this study is to assess the (cost)-effectiveness and feasibility of the MCI, aiming to improve self-efficacy and concordance in patients with epilepsy.

Methods

A RCT in two parallel groups will be conducted to compare the MCI with a control condition in epilepsy patients. One hundred eligible epilepsy patients will be recruited and allocated to either the intervention or control group. The intervention group will receive the MCI consisting of a self-management/education program of six meetings, including e-Health interventions, and will be followed for 12 months. The control group will receive care as usual and will be followed for 6 months, after which patients will be offered the possibility of participating in the MCI. The study will consist of three parts: 1) a clinical effectiveness study, 2) a cost-effectiveness study, and 3) process evaluation. The primary outcome will be self-efficacy. Secondary outcomes include adherence, side effects, change in seizure severity & frequency, improved quality of life, proactive coping, and societal costs. Outcome assessments will be done using questionnaires at baseline and after 3, 6, 9, and 12 months (last two applicable only for intervention group).

Discussion

In times of budget constraints, MCI could be a valuable addition to the current healthcare provision for epilepsy, as it is expected that higher concordance and self-efficacy will result in reduced use of healthcare resources and an increased QOL. Accordingly, this study is aimed helping patients to be their own provider of health care, shifting epilepsy management from professionals to self-care by patients equipped with appropriate skills and tools.

Trial registration number

NTR4484.

     

Glycosome turnover in Leishmania major is mediated by autophagy

Autophagy is a central process behind the cellular remodeling that occurs during differentiation of Leishmania, yet the cargo of the protozoan parasite''s autophagosome is unknown. We have identified glycosomes, peroxisome-like organelles that uniquely compartmentalize glycolytic and other metabolic enzymes in Leishmania and other kinetoplastid parasitic protozoa, as autophagosome cargo. It has been proposed that the number of glycosomes and their content change during the Leishmania life cycle as a key adaptation to the different environments encountered. Quantification of RFP-SQL-labeled glycosomes showed that promastigotes of L. major possess ∼20 glycosomes per cell, whereas amastigotes contain ∼10. Glycosome numbers were significantly greater in promastigotes and amastigotes of autophagy-defective L. major Δatg5 mutants, implicating autophagy in glycosome homeostasis and providing a partial explanation for the previously observed growth and virulence defects of these mutants. Use of GFP-ATG8 to label autophagosomes showed glycosomes to be cargo in ∼15% of them; glycosome-containing autophagosomes were trafficked to the lysosome for degradation. The number of autophagosomes increased 10-fold during differentiation, yet the percentage of glycosome-containing autophagosomes remained constant. This indicates that increased turnover of glycosomes was due to an overall increase in autophagy, rather than an upregulation of autophagosomes containing this cargo. Mitophagy of the single mitochondrion was not observed in L. major during normal growth or differentiation; however, mitochondrial remnants resulting from stress-induced fragmentation colocalized with autophagosomes and lysosomes, indicating that autophagy is used to recycle these damaged organelles. These data show that autophagy in Leishmania has a central role not only in maintaining cellular homeostasis and recycling damaged organelles but crucially in the adaptation to environmental change through the turnover of glycosomes.