Phosphorylcholine is favorable for antibody production from hybridoma cells

Growth of antibody-secreting hybridomas requires special conditions such as serum-free defined media containing growth factors and vitamins. However, the surface on which these cells can proliferate has been shown to play an important role. Phosphorylcholine (PC)-based polymers are zwitterionic compounds with nonbiofouling properties. These polymers are characterized by having reduced protein absorption properties. Our aim was to determine whether well-established hybridoma cell lines were able to proliferate and produce measurable amounts of monoclonal antibodies when grown on PC-polymer-coated surfaces. Comparative experiments using four well-known hybridoma cell lines (PAb421, PAb246, PAb1801 which recognize p53, and PAb280 which recognizes SV40 small t antigen) grown on PC-polymer-coated, uncoated, and two commercially available tissue culture plates showed that PC-polymer-coated plates were more efficient than uncoated plates in sustaining cell growth and monoclonal antibody production/secretion as defined by growth assays and ELISA. Also, results demonstrated that PC-polymer-coated plates were able to perform better than commercially available plates. These observations suggest that PC polymers could be used as an alternative, efficient surface coating to grow hybridoma cell lines and allow detectable antibody secretion.     

Signal transduction in lemon seedlings in the hypersensitive response against Alternaria alternata: participation of calmodulin,G-protein and protein kinases

The development of an effective hypersensitive response (HR) in any plant system relies, not only in their gene composition and expression, but also on an effective and rapid signal transduction system. Lemon seedlings induce the phenylpropanoid pathway, which results in the de novo biosynthesis of the phytoalexin scoparone, as part of the hypersensitive response against Alternaria alternata. In order to elucidate some of the signaling elements that participate in the development of HR in lemon seedlings, we used several compounds that are known as activators or inhibitors of signal transduction elements in plants or in animal cells. Lemon seedlings treated either with cholera toxin or with phorbol 12-myristate 13-acetate (PMA), in the absence of A. alternata induced phenylalanine ammonia-lyase (PAL, E. C. 4.3.1.5) and the synthesis of scoparone, suggesting the participation of a G-protein and of a serine/threonine kinase, respectively, in signal transduction. The use of trifluoperazine (TFP), W-7, staurosporine, lavendustin A or 2,5-dihydroximethyl cinnamate (DHMC) prevented PAL induction as well as scoparone biosynthesis in response to the fungal inoculation, thus allowing us to infer the participation of Calmodulin (CaM), of serine/threonine and of tyrosine protein kinases (TPK) for signal transduction in Citrus limon in response to A. alternata.     

Increasing the potency of neutralizing single-domain antibodies by functionalization with a CD11b/CD18 binding domain

Recombinant single domain antibodies (nanobodies) constitute an attractive alternative for the production of neutralizing therapeutic agents. Their small size warrants rapid bioavailability and fast penetration to sites of toxin uptake, but also rapid renal clearance, which negatively affects their performance. In this work, we present a new strategy to drastically improve the neutralizing potency of single domain antibodies based on their fusion to a second nanobody specific for the complement receptor CD11b/CD18 (Mac-1). These bispecific antibodies retain a small size (˜30 kDa), but acquire effector functions that promote the elimination of the toxin-immunocomplexes. The principle was demonstrated in a mouse model of lethal toxicity with tetanus toxin. Three anti-tetanus toxin nanobodies were selected and characterized in terms of overlapping epitopes and inhibition of toxin binding to neuron gangliosides. Bispecific constructs of the most promising monodomain antibodies were built using anti Mac-1, CD45 and MHC II nanobodies. When co-administered with the toxin, all bispecific antibodies showed higher toxin-neutralizing capacity than the monomeric ones, but only their fusion to the anti-endocytic receptor Mac-1 nanobody allowed the mice to survive a 10-fold lethal dose. In a model of delayed neutralization of the toxin, the anti- Mac-1 bispecific antibodies outperformed a sheep anti-toxin polyclonal IgG that had shown similar neutralization potency in the co-administration experiments. This strategy should have widespread application in the development of nanobody-based neutralizing therapeutics, which can be produced economically and more safely than conventional antisera.     

DNA-Methylation Patterns in Trisomy 21 Using Cells from Monozygotic Twins

DNA methylation is essential in mammalian development. We have hypothesized that methylation differences induced by trisomy 21 (T21) contribute to the phenotypic characteristics and heterogeneity in Down syndrome (DS). In order to determine the methylation differences in T21 without interference of the interindividual genomic variation, we have used fetal skin fibroblasts from monozygotic (MZ) twins discordant for T21. We also used skin fibroblasts from MZ twins concordant for T21, normal MZ twins without T21, and unrelated normal and T21 individuals. Reduced Representation Bisulfite Sequencing (RRBS) revealed 35 differentially methylated promoter regions (DMRs) (Absolute methylation differences = 25%, FDR < 0.001) in MZ twins discordant for T21 that have also been observed in comparison between unrelated normal and T21 individuals. The identified DMRs are enriched for genes involved in embryonic organ morphogenesis (FDR = 1.60 e -03) and include genes of the HOXB and HOXD clusters. These DMRs are maintained in iPS cells generated from this twin pair and are correlated with the gene expression changes. We have also observed an increase in DNA methylation level in the T21 methylome compared to the normal euploid methylome. This observation is concordant with the up regulation of DNA methyltransferase enzymes (DNMT3B and DNMT3L) and down regulation of DNA demethylation enzymes (TET2 and TET3) observed in the iPSC of the T21 versus normal twin. Altogether, the results of this study highlight the epigenetic effects of the extra chromosome 21 in T21 on loci outside of this chromosome that are relevant to DS associated phenotypes.     

Muscle-Derived Extracellular Signal-Regulated Kinases 1 and 2 Are Required for the Maintenance of Adult Myofibers and Their Neuromuscular Junctions

The Ras–extracellular signal-regulated kinase 1 and 2 (ERK1/2) pathway appears to be important for the development, maintenance, aging, and pathology of mammalian skeletal muscle. Yet no gene targeting of Erk1/2 in muscle fibers in vivo has been reported to date. We combined a germ line Erk1 mutation with Cre-loxP Erk2 inactivation in skeletal muscle to produce, for the first time, mice lacking ERK1/2 selectively in skeletal myofibers. Animals lacking muscle ERK1/2 displayed stunted postnatal growth, muscle weakness, and a shorter life span. Their muscles examined in this study, sternomastoid and tibialis anterior, displayed fragmented neuromuscular synapses and a mixture of modest fiber atrophy and loss but failed to show major changes in fiber type composition or absence of cell surface dystrophin. Whereas the lack of only ERK1 had no effects on the phenotypes studied, the lack of myofiber ERK2 explained synaptic fragmentation in the sternomastoid but not the tibialis anterior and a decrease in the expression of the acetylcholine receptor (AChR) epsilon subunit gene mRNA in both muscles. A reduction in AChR protein was documented in line with the above mRNA results. Evidence of partial denervation was found in the sternomastoid but not the tibialis anterior. Thus, myofiber ERK1/2 are differentially required for the maintenance of myofibers and neuromuscular synapses in adult mice.     

A garden experiment revisited: inter‐generational change in environmental perception and management of the Maya Lowlands,Guatemala

This study reports ethnographic and experimental analyses of inter‐generational changes in native Itza' Maya and immigrant Ladino populations of Guatemala's Petén rainforest concerning understanding of ecological relationships between plants, animals, and humans, and the perceived role of forest spirits in sustaining these relationships. We find dramatic changes in understanding ecological relationships and the perceived role of forest spirits. Itza' Maya conceptions of forest spirits (arux) are now more often confounded with Ladino spirits (duendes), with Itza' spirits no longer reliably serving as forest guardians. These changes correlate with a shift in personal values regarding the forest, away from concern with ecologically central trees and towards monetary incentives. More generally, we describe how economic, demographic, and social changes relate to the loss of a system of beliefs and behaviours that once promoted sustainable agro‐forestry practices. These changes coincide with open access to common pool resources.     

Archaea-based microbial fuel cell operating at high ionic strength conditions

In this work, two archaea microorganisms (Haloferax volcanii and Natrialba magadii) used as biocatalyst at a microbial fuel cell (MFC) anode were evaluated. Both archaea are able to grow at high salt concentrations. By increasing the media conductivity, the internal resistance was diminished, improving the MFC’s performance. Without any added redox mediator, maximum power (P _max) and current at P _max were 11.87/4.57/0.12 μW cm⁻² and 49.67/22.03/0.59 μA cm⁻² for H. volcanii, N. magadii and E. coli, respectively. When neutral red was used as the redox mediator, P _max was 50.98 and 5.39 μW cm⁻² for H. volcanii and N. magadii, respectively. In this paper, an archaea MFC is described and compared with other MFC systems; the high salt concentration assayed here, comparable with that used in Pt-catalyzed alkaline hydrogen fuel cells, will open new options when MFC scaling up is the objective necessary for practical applications.     

Elimination of neglected diseases in latin america and the Caribbean: a mapping of selected diseases

In Latin America and the Caribbean, around 195 million people live in poverty, a situation that increases the burden of some infectious diseases. Neglected diseases, in particular, are often restricted to poor, marginalized sections of the population. Tools exist to combat these diseases, making it imperative to work towards their elimination. In 2009, the Pan American Health Organization (PAHO) received a mandate to support the countries in the Region in eliminating neglected diseases and other poverty-related infections. The objective of this study is to analyze the presence of selected diseases using geo-processing techniques. Five diseases with information available at the first sub-national level (states) were mapped, showing the presence of the disease ("hotspots") and overlap of diseases ("major hotspots"). In the 45 countries/territories (approximately 570 states) of the Region, there is: lymphatic filariasis in four countries (29 states), onchocerciasis in six countries (25 states), schistosomiasis in four countries (39 states), trachoma in three countries (29 states), and human rabies transmitted by dogs in ten countries (20 states). Of the 108 states with one or more of the selected diseases, 36 states present the diseases in overlapping areas ("major hotspots"). Additional information about soil-transmitted helminths was included. The analysis suggests a majority of the selected diseases are not widespread and can be considered part of an unfinished agenda with elimination as a goal. Integrated plans and a comprehensive approach, ensuring access to existing diagnostic and treatment methods, and establishing a multi-sectoral agenda that addresses social determinants, including access to adequate water and sanitation, are required. Future studies can include additional diseases, socio-economic and environmental variables.