Involvement of ethylene and nitric oxide in cell death in mastoparan‐treated unicellular alga Chlamydomonas reinhardtii

This work demonstrates a contribution of ethylene and NO (nitric oxide) in MP (mastoparan)‐induced cell death in the green algae Chlamydomonas reinhardtii. Following MP treatment, C. reinhardtii showed massive cell death, expressing morphological features of PCD (programmed cell death). A pharmacological approach involving combined treatments with MP and ethylene‐ and NO‐interacting compounds indicated the requirement of trace amounts of both ethylene and NO in MP‐induced cell death. By employing a carbon dioxide laser‐based photoacoustic detector to measure ethylene and a QCL (quantum cascade laser)‐based spectrometer for NO detection, simultaneous increases in the production of both ethylene and NO were observed following MP application. Our results show a tight regulation of the levels of both signalling molecules in which ethylene stimulates NO production and NO stimulates ethylene production. This suggests that, in conjunction with the elicitor, NO and ethylene cooperate and act synchronously in the mediation of MP‐induced PCD in C. reinhardtii. To the best of our knowledge, this is the first report on the functional significance of ethylene and NO in MP‐induced cell death.     

Plant‐insect interactions: what can we learn from plant lectins?

Many plant lectins have high anti‐insect potential. Although the effects of most lectins are only moderately influencing development or population growth of the insect, some lectins have strong insecticidal properties. In addition, some studies report a deterrent activity towards feeding and oviposition behavior. Transmission of plant lectins to the next trophic level has been investigated for several tritrophic interactions. Effects of lectins with different sugar specificities can vary substantially with the insect species under investigation and with the experimental setup. Lectin binding in the insect is an essential step in exerting a toxic effect. Attempts have been made to study the interactions of lectins in several insect tissues and to identify lectin‐binding receptors. Ingested lectins generally bind to parts of the insect gut. Furthermore, some lectins such as the Galanthus nivalus agglutinin (GNA) cross the gut epithelium into the hemolymph and other tissues. Recently, several candidate lectin‐binding receptors have been isolated from midgut extracts. To date little is known about the exact mechanism for insecticidal activity of plant lectins. However, insect glycobiology is an emerging research field and the recent technological advances in the analysis of lectin carbohydrate specificities and insect glycobiology will certainly lead to new insights in the interactions between plant lectins and insects, and to a better understanding of the molecular mechanisms involved. © 2010 Wiley Periodicals, Inc.     

CD97 neutralisation increases resistance to collagen-induced arthritis in mice

Synovial tissue of rheumatoid arthritis (RA) patients is characterised by an influx and retention of CD97-positive inflammatory cells. The ligands of CD97, CD55, chondroitin sulfate B, and α5β1 (very late antigen [VLA]-5) are expressed abundantly in the synovial tissue predominantly on fibroblast-like synoviocytes, endothelium, and extracellular matrix. Based upon this expression pattern, we hypothesise CD97 expression to result in accumulation of inflammatory cells in the synovial tissue of RA patients. To determine the therapeutic effect of blocking CD97 in an animal model of RA, collagen-induced arthritis was induced in a total of 124 DBA/J1 mice. Treatment was started on day 21 (early disease) or on day 35 (longstanding disease) with the blocking hamster anti-mouse CD97 monoclonal antibody (mAb) 1B2, control hamster immunoglobulin, or NaCl, applied intraperitoneally three times a week. The paws were evaluated for clinical signs of arthritis and, in addition, examined by radiological and histological analysis. Mice receiving 0.5 mg CD97 mAb starting from day 21 had significantly less arthritis activity and hind paw swelling. Furthermore, joint damage and inflammation were reduced and granulocyte infiltration was decreased. When treatment was started on day 35, CD97 mAb treatment had similar effects, albeit less pronounced. The results support the notion that CD97 contributes to synovial inflammation and joint destruction in arthritis.     

Deployment of ACT antimalarials for treatment of malaria: challenges and opportunities

Background

In late 2002, the health authorities of Mozambique implemented sulphadoxine-pyrimethamine (SP)/amodiaquine (AQ) as first-line treatment against uncomplicated falciparum malaria. In 2004, this has been altered to SP/artesunate in line with WHO recommendations of using Artemisinin Combination Therapies (ACTs), despite the fact that all the neighbouring countries have abandoned SP-drug combinations due to high levels of SP drug resistance. In the study area, one year prior to the change to SP/AQ, SP alone was used to treat uncomplicated malaria cases. The study described here investigated the immediate impact of the change to SP on the frequency of SP and CQ resistance-related haplotypes in the Plasmodium falciparum genes Pfdhfr, Pfdhps and Pfcrt before and a year after the introduction of SP.

Methods

Samples were collected during two cross sectional surveys in early 2002 and 2003 involving 796 and 692 children one year or older and adults randomly selected living in Maciana, an area located in Manhiça district, Southern Mozambique. Out of these, 171 and 173 P. falciparum positive samples were randomly selected to measure the frequency of resistance- related haplotypes in Pfdhfr, Pfdhps and Pfcrt based on results obtained by nested PCR followed by sequence-specific oligonucleotide probe (SSOP)-ELISA.

Results

The frequency of the SP-resistance associated Pfdhps double mutant (SGEAA) haplotype increased significantly from 14% to 35% (P < 0.001), while the triple mutant Pfdhfr haplotype (CIRNI) remained high and only changed marginally from 46% to 53% (P = 0.405) after one year with SP as first-line treatment in the study area. Conversely, the combined Pfdhfr/Pfdhps quintuple mutant haplotype increased from 8% to 26% (P = 0.005). The frequency of the chloroquine resistance associated Pfcrt -CVIET haplotype was above 90% in both years.

Conclusion

These retrospective findings add to the general concern on the lifespan of the combination of SP/artesunate in Mozambique. The high frequency of quintuple Pfdhfr/Pfdhps haplotypes found here as early as 2002 most likely cause ideal conditions for the development of artesunate tolerance in the P. falciparum populations and may even endanger the sensitivity to the second-line drug, Coartem^®.     

Non-coding-regulatory regions of human brain genes delineated by bacterial artificial chromosome knock-in mice

Background

The next big challenge in human genetics is understanding the 98% of the genome that comprises non-coding DNA. Hidden in this DNA are sequences critical for gene regulation, and new experimental strategies are needed to understand the functional role of gene-regulation sequences in health and disease. In this study, we build upon our HuGX ('high-throughput human genes on the X chromosome’) strategy to expand our understanding of human gene regulation in vivo.

Results

In all, ten human genes known to express in therapeutically important brain regions were chosen for study. For eight of these genes, human bacterial artificial chromosome clones were identified, retrofitted with a reporter, knocked single-copy into the Hprt locus in mouse embryonic stem cells, and mouse strains derived. Five of these human genes expressed in mouse, and all expressed in the adult brain region for which they were chosen. This defined the boundaries of the genomic DNA sufficient for brain expression, and refined our knowledge regarding the complexity of gene regulation. We also characterized for the first time the expression of human MAOA and NR2F2, two genes for which the mouse homologs have been extensively studied in the central nervous system (CNS), and AMOTL1 and NOV, for which roles in CNS have been unclear.

Conclusions

We have demonstrated the use of the HuGX strategy to functionally delineate non-coding-regulatory regions of therapeutically important human brain genes. Our results also show that a careful investigation, using publicly available resources and bioinformatics, can lead to accurate predictions of gene expression.

     

Searching for phenotypic causal networks involving complex traits: an application to European quail

Background

Structural equation models (SEM) are used to model multiple traits and the casual links among them. The number of different causal structures that can be used to fit a SEM is typically very large, even when only a few traits are studied. In recent applications of SEM in quantitative genetics mixed model settings, causal structures were pre-selected based on prior beliefs alone. Alternatively, there are algorithms that search for structures that are compatible with the joint distribution of the data. However, such a search cannot be performed directly on the joint distribution of the phenotypes since causal relationships are possibly masked by genetic covariances. In this context, the application of the Inductive Causation (IC) algorithm to the joint distribution of phenotypes conditional to unobservable genetic effects has been proposed.

Methods

Here, we applied this approach to five traits in European quail: birth weight (BW), weight at 35 days of age (W35), age at first egg (AFE), average egg weight from 77 to 110 days of age (AEW), and number of eggs laid in the same period (NE). We have focused the discussion on the challenges and difficulties resulting from applying this method to field data. Statistical decisions regarding partial correlations were based on different Highest Posterior Density (HPD) interval contents and models based on the selected causal structures were compared using the Deviance Information Criterion (DIC). In addition, we used temporal information to perform additional edge orienting, overriding the algorithm output when necessary.

Results

As a result, the final causal structure consisted of two separated substructures: BW→AEW and W35→AFE→NE, where an arrow represents a direct effect. Comparison between a SEM with the selected structure and a Multiple Trait Animal Model using DIC indicated that the SEM is more plausible.

Conclusions

Coupling prior knowledge with the output provided by the IC algorithm allowed further learning regarding phenotypic causal structures when compared to standard mixed effects SEM applications.     

Analysis of the genomic response of human prostate cancer cells to histone deacetylase inhibitors

Histone deacetylases (HDACs) have emerged as important targets for cancer treatment. HDAC-inhibitors (HDACis) are well tolerated in patients and have been approved for the treatment of patients with cutaneous T-cell lymphoma (CTCL). To improve the clinical benefit of HDACis in solid tumors, combination strategies with HDACis could be employed. In this study, we applied Analysis of Functional Annotation (AFA) to provide a comprehensive list of genes and pathways affected upon HDACi-treatment in prostate cancer cells. This approach provides an unbiased and objective approach to high throughput data mining. By performing AFA on gene expression data from prostate cancer cell lines DU-145 (an HDACi-sensitive cell line) and PC3 (a relatively HDACi-resistant cell line) treated with HDACis valproic acid or vorinostat, we identified biological processes that are affected by HDACis and are therefore potential treatment targets for combination therapy. Our analysis revealed that HDAC-inhibition resulted among others in upregulation of major histocompatibility complex (MHC) genes and deregulation of the mitotic spindle checkpoint by downregulation of genes involved in mitosis. These findings were confirmed by AFA on publicly available data sets from HDACi-treated prostate cancer cells. In total, we analyzed 375 microarrays with HDACi treated and non-treated (control) prostate cancer cells. All results from this extensive analysis are provided as an online research source (available at the journal’s website and at http://luigimarchionni.org/HDACIs.html). By publishing this data, we aim to enhance our understanding of the cellular changes after HDAC-inhibition, and to identify novel potential combination strategies with HDACis for the treatment of prostate cancer patients.     

CLARIFYING APPEALS TO DIGNITY IN MEDICAL ETHICS FROM AN HISTORICAL PERSPECTIVE

Over the past few decades the concept of (human) dignity has deeply pervaded medical ethics. Appeals to dignity, however, are often unclear. As a result some prefer to eliminate the concept from medical ethics, whereas others try to render it useful in this context. We think that appeals to dignity in medical ethics can be clarified by considering the concept from an historical perspective. Firstly, on the basis of historical texts we propose a framework for defining the concept in medical debates. The framework shows that dignity can occur in a relational, an unconditional, a subjective and a Kantian form. Interestingly, all forms relate to one concept since they have four features in common: dignity refers, in a restricted sense, to the 'special status of human beings'; it is based on essential human characteristics; the subject of dignity should live up to it; and it is a vulnerable concept, it can be lost or violated. We argue that being explicit about the meaning of dignity will prevent dignity from becoming a conversation-stopper in moral debate. Secondly, an historical perspective on dignity shows that it is not yet time to dispose of dignity in medical ethics. At least Kantian and relational dignity can be made useful in medical ethics.     

Statistical evidence for two major proteins in freeze-fractured gastric parietal cell tubulovesicles and canaliculus

In a previous work, resting and acid-secreting rabbit gastric mucosa were freeze-fractured and shadowed at 45° with Pt-C. The shadow widths of proteic particles of tubulovesicle and canaliculus membranes were measured and compared. It was concluded that the frequency distributions of widths are significantly different in resting and secreting membranes and that each distribution accounts for several subpopulations of homogenous particles. In the present study, an attempt is made to describe the experimental distributions as a mixture of those of two major proteins, say A and B and their aggregates (AA, AB and BB). The modelling, although simple, gave a very satisfactory statistical fit between observed and computed distributions. The comparison of parameters calculated from histamine and ranitidine experimental data further improves the fits and finally, component A accounts for 69% of the particles. Most replica of A particles are heart-shaped and the median shadow widths are 6.1 and 6.8 nm in canaliculus and tubulovesicles respectively. The component B accounts for 31% of the particles. They mainly appear as small barrels and the median shadow widths are 8.8 and 10.3 nm in canaliculus and tubulovesicles respectively. According to calculated parameters and observed particle replica, the onset of secretion does not change the relative ratio of proteins but changes their shapes. Component A should be the (H⁺, K⁺)ATPase whereas debate on the identity of B is wide open.