Evaluation of chloroform/methanol extraction to facilitate the study of membrane proteins of non-model plants

Membrane proteins are of great interest to plant physiologists because of their important function in many physiological processes. However, their study is hampered by their low abundance and poor solubility in aqueous buffers. Proteomics studies of non-model plants are generally restricted to gel-based methods. Unfortunately, all gel-based techniques for membrane proteomics lack resolving power. Therefore, a very stringent enrichment method is needed before protein separation. In this study, protein extraction in a mixture of chloroform and methanol in combination with gel electrophoresis is evaluated as a method to study membrane proteins in non-model plants. Benefits as well as disadvantages of the method are discussed. To demonstrate the pitfalls of working with non-model plants and to give a proof of principle, the method was first applied to whole leaves of the model plant Arabidopsis. Subsequently, a comparison with proteins extracted from leaves of the non-model plant, banana, was made. To estimate the tissue and organelle specificity of the method, it was also applied on banana meristems. Abundant membrane or lipid-associated proteins could be identified in both tissues, with the leaf extract yielding a higher number of membrane proteins.     

Using seminatural and simulated habitats for seed germination ecology of banana wild relatives

1. Ecologically meaningful seed germination experiments are constrained by access to seeds and relevant environments for testing at the same time. This is particularly the case when research is carried out far from the native area of the studied species.
2. Here, we demonstrate an alternative—the use of glasshouses in botanic gardens as simulated‐natural habitats to extend the ecological interpretation of germination studies. Our focal taxa were banana crop wild relatives (Musa acuminata subsp. burmannica, Musa acuminata subsp. siamea, and Musa balbisiana), native to tropical and subtropical South‐East Asia. Tests were carried out in Belgium, where we performed germination tests in relation to foliage‐shading/exposure to solar radiation and seed burial depth, as well as seed survival and dormancy release in the soil. We calibrated the interpretation of these studies by also conducting an experiment in a seminatural habitat in a species native range (M. balbisiana—Los Baños, the Philippines), where we tested germination responses to exposure to sun/shade. Using temperature data loggers, we determined temperature dynamics suitable for germination in both these settings.
3. In these seminatural and simulated‐natural habitats, seeds germinated in response to exposure to direct solar radiation. Seed burial depth had a significant but marginal effect by comparison, even when seeds were buried to 7 cm in the soil. Temperatures at sun‐exposed compared with shaded environments differed by only a few degrees Celsius. Maximum temperature of the period prior to germination was the most significant contributor to germination responses and germination increased linearly above a threshold of 23℃ to the maximum temperature in the soil (in simulated‐natural habitats) of 35℃.
4. Glasshouses can provide useful environments to aid interpretation of seed germination responses to environmental niches.

     

First archaeological evidence of banana cultivation in central Africa during the third millennium before present

Phytoliths recovered from refuse pits excavated in central Cameroon and dated to ca 2500 B.P. have been positively identified for the first time in Africa as derived from Musa the cultivated banana, after a comparative study of Musa and Ensete phytoliths. This discovery provides archaeologists with unequivocal proof of early agriculture in central Africa. Furthermore, the presence of banana in Cameroon much earlier than previously assumed could explain how agriculture spread through the rain forest. Lastly, as Musa is of Asian origin, this study provides the first concrete evidence of contacts across the Indian Ocean a millennium earlier than currently accepted. Received July 12, 1999 / Accepted May 4, 2000     

Coexistence of Flexibility and Stability of Proteins: An Equation of State

We consider a recently suggested “equation of state” for natively folded proteins, and verify its validity for a set of about 5800 proteins. The equation is based on a fractal viewpoint of proteins, on a generalization of the Landau-Peierls instability, and on a marginal stability criterion. The latter allows for coexistence of stability and flexibility of proteins, which is required for their proper function. The equation of state relates the protein fractal dimension , its spectral dimension , and the number of amino acids N. Using structural data from the protein data bank (PDB) and the Gaussian network model (GNM), we compute and for the entire set and demonstrate that the equation of state is well obeyed. Addressing the fractal properties and making use of the equation of state may help to engineer biologically inspired catalysts.     

Neglect of a Neglected Disease in Italy: The Challenge of Access-to-Care for Chagas Disease in Bergamo Area

ObjectivesChagas disease (CD) represents a growing problem in Europe; Italy is one of the most affected countries but there is no national framework for CD and access-to-care is challenging. In 2012 Médecins Sans Frontières (MSF) started an intervention in Bergamo province, where many people of Latin American origin (PLAO) are resident. A new model-of-care for CD, initiated by Centre for Tropical Diseases of Sacro Cuore Hospital, Negrar (CTD), the NGO OIKOS and the Bolivian community since 2009 in the same area, was endorsed. Hereby, we aim to describe the prevalence of CD and the treatment management outcomes among PLAO screened from 1^st June 2012 to 30^th June 2013.MethodsRetrospective cohort study using routine program data. Screening sessions were done in Bergamo at OIKOS outpatient service and serological confirmation, staging and treatment for CD was offered at the CTD. MSF provided health education on CD, awareness generation prior to screening days, pre-test and post-test counselling through cultural mediators of Latin American origin.ResultsOf 1305 PLAO screened, 223(17%) had CD. Among 210 patients eligible for treatment, 102(49%) were lost-to-follow-up before treatment. The median delay from diagnosis to treatment was 4 months (range 0.7–16.6 months). Among 108 started on treatment, 63(58%) completed treatment, 36(33%) interrupted treatment, (33 for drug side-effects, two for patients decision and one due to pregnancy), 6(6%) were lost-to-follow-up and 3(3%) were on treatment at study censuring.ConclusionIn this first study focusing on process of care for CD in Italy, less than 30% of patients completed treatment with drop-outs along the cascade of care. There is an urgent need to involve affected communities and local regional health authorities to take part to this model-of-care, adapting it to the local epidemiology. The Italian health authorities should take steps in advocating for a change in the current paradigm.     

Molecular and Cytogenetic Characterization of Wild Musa Species

The production of bananas is threatened by rapid spreading of various diseases and adverse environmental conditions. The preservation and characterization of banana diversity is essential for the purposes of crop improvement. The world''s largest banana germplasm collection maintained at the Bioversity International Transit Centre (ITC) in Belgium is continuously expanded by new accessions of edible cultivars and wild species. Detailed morphological and molecular characterization of the accessions is necessary for efficient management of the collection and utilization of banana diversity. In this work, nuclear DNA content and genomic distribution of 45S and 5S rDNA were examined in 21 diploid accessions recently added to ITC collection, representing both sections of the genus Musa. 2C DNA content in the section Musa ranged from 1.217 to 1.315 pg. Species belonging to section Callimusa had 2C DNA contents ranging from 1.390 to 1.772 pg. While the number of 45S rDNA loci was conserved in the section Musa, it was highly variable in Callimusa species. 5S rRNA gene clusters were found on two to eight chromosomes per diploid cell. The accessions were genotyped using a set of 19 microsatellite markers to establish their relationships with the remaining accessions held at ITC. Genetic diversity done by SSR genotyping platform was extended by phylogenetic analysis of ITS region. ITS sequence data supported the clustering obtained by SSR analysis for most of the accessions. High level of nucleotide diversity and presence of more than two types of ITS sequences in eight wild diploids pointed to their origin by hybridization of different genotypes. This study significantly expands the number of wild Musa species where nuclear genome size and genomic distribution of rDNA loci is known. SSR genotyping identified Musa species that are closely related to the previously characterized accessions and provided data to aid in their classification. Sequence analysis of ITS region provided further information about evolutionary relationships between individual accessions and suggested that some of analyzed accessions were interspecific hybrids and/or backcross progeny.     

Electromagnetic Interference in a Private Swimming Pool: Case report

Although current lead design and filtering capabilities have greatly improved, Electromagnetic Interference (EMI) from environmental sources has been increasingly reported in patients with Cardiac Implantable Electronic Device (CIED) [1]. Few cases of inappropriate intracardiac Cardioverter Defibrillator (ICD) associated with swimming pool has been described [2]. Here we present a case of 64 year old male who presented with an interesting EMI signal that was subsequently identified to be related to AC current leak in his swimming pool.     

Gq protein-induced apoptosis is mediated by AKT kinase inhibition that leads to protein kinase C-induced c-Jun N-terminal kinase activation

G(q) protein-coupled receptors (G(q)PCRs) regulate various cellular processes, including mainly proliferation and differentiation. In a previous study we found that in prostate cancer cells, the G(q)PCR of gonadotropin-releasing hormone (GnRH) induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Because it was thought that G(q)PCRs mainly induce activation of AKT, we first undertook to examine how general this phenomenon is. In a screen of 21 cell lines we found that PKC activation results in the reduction of AKT activity, which correlates nicely with JNK activation and in some cases with apoptosis. To understand further the signaling pathways involved in this stimulation, we studied in detail SVOG-4O and αT3-1 cells. We found that prostaglandin F2α and GnRH agonist (GnRH-a) indeed induce significant Gα(q)- and PKC-dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists of c-Src activation of the JNK cascade, and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3 to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Our results present a general mechanism that mediates a G(q)PCR-induced, death receptor-independent, apoptosis in physiological, as well as cancer-related systems.