A rapid double-staining technique to improve seed viability testing in terrestrial orchids

Abstract

The need to optimize seed banking efforts has stimulated research for rapid methods to estimate quality in seed-lots. For terrestrial orchids, viability testing using tetrazolium (TTC) staining requires chemical scarification, as seeds have an impermeable testa. Different seed-coat permeability may affect TTC staining, thus affecting the results. The aim of this study was to perform a permeability test to assess the effectiveness of the used scarification method and its usefulness to correct TTC viability results. Mature seeds of Anacamptis laxiflora were subjected to eight scarification treatments with sodium hypochlorite solutions with different concentration and duration. Viability tests were performed using the basic TTC methodology, followed by a permeability test performed by means of trypan blue dye. The different scarification methods resulted in estimated TTC viability ranging from 0% and 94% for the same seed lot of A. laxiflora seeds. Our results proved that the used scarification protocol significantly affects both seed coat permeability and subsequent TTC staining (two-way ANOVA, p?< 0.0001). We describe a new rapid protocol that can be used to test terrestrial orchid seed viability. This double-staining method, providing rapid information on seed coat permeability, can be useful to avoid under-estimation of TTC results.     

Three new hybrids of Ophioglossum (Ophioglossaceae) from Monte Pisano,Tuscany (Central Italy)

Based on general morphology, spore measurements and ornamentation (scanning electron microscope), genome size estimation, and molecular systematics (trnL-trnF IGS), we show the extreme systematic complexity within the European representatives of the genus Ophioglossum. In particular, three hybrids from Tuscany are described: the tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane, seen as the hybrid between diploid O. lusitanicum L. and hexaploid O. azoricum C.Presl; the tetraploid O. × giovanninii Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the homoploid hybrid between tetraploid O. vulgatum L. and tetraploid O. × pierinii Peruzzi, Magrini, Marchetti & Viane; the pentaploid O. × pseudoazoricum Peruzzi, Pierini, Magrini, Marchetti & Viane, seen as the hybrid between hexaploid O. azoricum C.Presl and tetraploid O. vulgatum L. All the three new taxa grow in different localities in the Monte Pisano mountain range.     

The Oxytricha trifallax Macronuclear Genome: A Complex Eukaryotic Genome with 16,000 Tiny Chromosomes

The macronuclear genome of the ciliate Oxytricha trifallax displays an extreme and unique eukaryotic genome architecture with extensive genomic variation. During sexual genome development, the expressed, somatic macronuclear genome is whittled down to the genic portion of a small fraction (∼5%) of its precursor “silent” germline micronuclear genome by a process of “unscrambling” and fragmentation. The tiny macronuclear “nanochromosomes” typically encode single, protein-coding genes (a small portion, 10%, encode 2–8 genes), have minimal noncoding regions, and are differentially amplified to an average of ∼2,000 copies. We report the high-quality genome assembly of ∼16,000 complete nanochromosomes (∼50 Mb haploid genome size) that vary from 469 bp to 66 kb long (mean ∼3.2 kb) and encode ∼18,500 genes. Alternative DNA fragmentation processes ∼10% of the nanochromosomes into multiple isoforms that usually encode complete genes. Nucleotide diversity in the macronucleus is very high (SNP heterozygosity is ∼4.0%), suggesting that Oxytricha trifallax may have one of the largest known effective population sizes of eukaryotes. Comparison to other ciliates with nonscrambled genomes and long macronuclear chromosomes (on the order of 100 kb) suggests several candidate proteins that could be involved in genome rearrangement, including domesticated MULE and IS1595-like DDE transposases. The assembly of the highly fragmented Oxytricha macronuclear genome is the first completed genome with such an unusual architecture. This genome sequence provides tantalizing glimpses into novel molecular biology and evolution. For example, Oxytricha maintains tens of millions of telomeres per cell and has also evolved an intriguing expansion of telomere end-binding proteins. In conjunction with the micronuclear genome in progress, the O. trifallax macronuclear genome will provide an invaluable resource for investigating programmed genome rearrangements, complementing studies of rearrangements arising during evolution and disease.     

Isolation, Characterization and Antifungal Activity of Proteinase Inhibitors from Capsicum chinense Jacq. Seeds

Capsicum species belong to the Solanaceae family and have great social, economic and agronomical significance. The present research presents data on the isolation and characterization of Capsicum chinense Jacq. peptides which were scrutinized in relation to their toxicity towards a diverse set of yeast species. The protein extract was separated with C18 reverse-phase chromatography in high performance liquid chromatography, resulting in three different peptide enriched fractions (PEFs) termed PEF1, PEF2 and PEF3. Tricine-SDS-PAGE of the PEF2 revealed peptides with molecular masses of approximately 5.0 and 8.5 kDa. These PEFs also exhibited strong antifungal activity against different yeasts. In the presence of the PEF2, Candida tropicalis exhibited morphological changes, including cellular agglomeration and formation of pseudohyphae. Determined N-terminal sequences of PEF2 and PEF3 were proven to be highly homologous to serine proteinase inhibitors, when analysed by comparative database sequence tools. For this reason were performed protease inhibitory activity assay. The PEFs displayed high inhibitory activity against trypsin and low inhibitory activity against chymotrypsin. PEF2 and PEF3 were considerably unsusceptible to a broad interval of pH and temperatures. Due to the myriad of application of Proteinase inhibitors (PIs) in fields ranging from plant protection against pathogens and pests to medicine such as in cancer and virus replication inhibition, the discovery of new PIs with new properties are of great interest.     

Traditional Nets Interfere with the Uptake of Long-Lasting Insecticidal Nets in the Peruvian Amazon: The Relevance of Net Preference for Achieving High Coverage and Use

Background

While coverage of long-lasting insecticide-treated nets (LLIN) has steadily increased, a growing number of studies report gaps between net ownership and use. We conducted a mixed-methods social science study assessing the importance of net preference and use after Olyset® LLINs were distributed through a mass campaign in rural communities surrounding Iquitos, the capital city of the Amazonian region of Peru.

Methods

The study was conducted in the catchment area of the Paujil and Cahuide Health Centres (San Juan district) between July 2007 and November 2008. During a first qualitative phase, participant observation and in-depth interviews collected information on key determinants for net preference and use. In a second quantitative phase, a survey among recently confirmed malaria patients evaluated the acceptability and use of both LLINs and traditional nets, and a case control study assessed the association between net preference/use and housing structure (open vs. closed houses).

Results

A total of 10 communities were selected for the anthropological fieldwork and 228 households participated in the quantitative studies. In the study area, bed nets are considered part of the housing structure and are therefore required to fulfil specific architectural and social functions, such as providing privacy and shelter, which the newly distributed Olyset® LLINs ultimately did not. The LLINs'' failure to meet these criteria could mainly be attributed to their large mesh size, transparency and perceived ineffectiveness to protect against mosquitoes and other insects, resulting in 63.3% of households not using any of the distributed LLINs. Notably, LLIN usage was significantly lower in houses with no interior or exterior walls (35.2%) than in those with walls (73.8%) (OR = 5.2, 95CI [2.2; 12.3], p<0.001).

Conclusion

Net preference can interfere with optimal LLIN use. In order to improve the number of effective days of LLIN protection per dollar spent, appropriate quantitative and qualitative methods for collecting information on net preference should be developed before any LLIN procurement decision is made.     

Cost-Utility Analysis of Lopinavir/Ritonavir versus Atazanavir + Ritonavir Administered as First-Line Therapy for the Treatment of HIV Infection in Italy: From Randomised Trial to Real World

Objective

To estimate the lifetime cost utility of two antiretroviral regimens (once-daily atazanavir plus ritonavir [ATV+r] versus twice-daily lopinavir/ritonavir [LPV/r]) in Italian human immunodeficiency virus (HIV)-infected patients naïve to treatment.

Design

With this observational retrospective study we collected the clinical data of a cohort of HIV-infected patients receiving first-line treatment with LPV/r or ATV+r.

Methodology

A Markov microsimulation model including direct costs and health outcomes of first- and second-line highly active retroviral therapy was developed from a third-party (Italian National Healthcare Service) payer’s perspective. Health and monetary outcomes associated with the long-term use of ATV+r and LPV/r regimens were evaluated on the basis of eight health states, incidence of diarrhoea and hyperbilirubinemia, AIDS events, opportunistic infections, coronary heart disease events and, for the first time in an economic evaluation, chronic kidney disease (CKD) events. In order to account for possible deviations between real-life data and randomised controlled trial results, a second control arm (ATV+r 2) was created with differential transition probabilities taken from the literature.

Results

The average survival was 24.061 years for patients receiving LPV/r, 24.081 and 24.084 for those receiving ATV+r 1 and 2 respectively. The mean quality-adjusted life-years (QALYs) were higher for the patients receiving LPV/r than those receiving ATV+r (13.322 vs. 13.060 and 13.261 for ATV+r 1 and 2). The cost-utility values were 15,310.56 for LPV/r, 15,902.99 and 15,524.85 for ATV+r 1 and 2.

Conclusions

Using real-life data, the model produced significantly different results compared with other studies. With the innovative addition of an evaluation of CKD events, the model showed a cost-utility value advantage for twice-daily LPV/r over once-daily ATV+r, thus providing evidence for its continued use in the treatment of HIV.     

Early and Middle Holocene Hunter-Gatherer Occupations in Western Amazonia: The Hidden Shell Middens

We report on previously unknown early archaeological sites in the Bolivian lowlands, demonstrating for the first time early and middle Holocene human presence in western Amazonia. Multidisciplinary research in forest islands situated in seasonally-inundated savannahs has revealed stratified shell middens produced by human foragers as early as 10,000 years ago, making them the oldest archaeological sites in the region. The absence of stone resources and partial burial by recent alluvial sediments has meant that these kinds of deposits have, until now, remained unidentified. We conducted core sampling, archaeological excavations and an interdisciplinary study of the stratigraphy and recovered materials from three shell midden mounds. Based on multiple lines of evidence, including radiocarbon dating, sedimentary proxies (elements, steroids and black carbon), micromorphology and faunal analysis, we demonstrate the anthropogenic origin and antiquity of these sites. In a tropical and geomorphologically active landscape often considered challenging both for early human occupation and for the preservation of hunter-gatherer sites, the newly discovered shell middens provide evidence for early to middle Holocene occupation and illustrate the potential for identifying and interpreting early open-air archaeological sites in western Amazonia. The existence of early hunter-gatherer sites in the Bolivian lowlands sheds new light on the region’s past and offers a new context within which the late Holocene “Earthmovers” of the Llanos de Moxos could have emerged.     

Electrophysiological characterization of the Cyclophilin D-deleted mitochondrial permeability transition pore

Mitochondria isolated from engineered mice lacking Cyclophilin D (CypD), a component of the Permeability Transition Pore (PTP) complex, can still undergo a Ca^2?+?-dependent but Cyclosporin A-insensitive permeabilization of the inner membrane. Higher Ca^2?+? concentrations are required than for wild-type controls. The characteristics of the pore formed in this system were not known, and it has been proposed that they might differ substantially from those of the normal PTP. To test this hypothesis, we have characterized the PTP of isogenic wild-type and CypD^? mouse liver mitochondria in patch clamp experiments, which allow biophysical characterization. The pores observed in the two cases, very similar to those of rat liver mitochondria, are indistinguishable according to a number of criteria. The only clear difference is in their sensitivity to Cyclosporin A. CypD is thus shown to be an auxiliary, modulatory component of the “standard” PTP, which forms and has essentially the same properties even in its absence. The observations suggest that Ca^2?+?, CypD, and presumably other inducers and inhibitors act at the level of an activation or assembly process. Activation is separate and upstream of the gating observable on a short or medium-term time scale. Once the pore is activated, its molecular dynamics and biophysical properties may thus be predicted not to depend on the details of the induction process.     

Stevens-Johnson Syndrome Associated with Drugs and Vaccines in Children: A Case-Control Study

Objective

Stevens-Johnson Syndrome (SJS) is one of the most severe muco-cutaneous diseases and its occurrence is often attributed to drug use. The aim of the present study is to quantify the risk of SJS in association with drug and vaccine use in children.

Methods

A multicenter surveillance of children hospitalized through the emergency departments for acute conditions of interest is currently ongoing in Italy. Cases with a diagnosis of SJS were retrieved from all admissions. Parents were interviewed on child’s use of drugs and vaccines preceding the onset of symptoms that led to the hospitalization. We compared the use of drugs and vaccines in cases with the corresponding use in a control group of children hospitalized for acute neurological conditions.

Results

Twenty-nine children with a diagnosis of SJS and 1,362 with neurological disorders were hospitalized between 1^st November 1999 and 31^st October 2012. Cases were more frequently exposed to drugs (79% vs 58% in the control group; adjusted OR 2.4; 95% CI 1.0–6.1). Anticonvulsants presented the highest adjusted OR: 26.8 (95% CI 8.4–86.0). Significantly elevated risks were also estimated for antibiotics use (adjusted OR 3.3; 95% CI 1.5–7.2), corticosteroids (adjusted OR 4.2; 95% CI 1.8–9.9) and paracetamol (adjusted OR 3.2; 95% CI 1.5–6.9). No increased risk was estimated for vaccines (adjusted OR: 0.9; 95% CI 0.3–2.8).

Discussion

Our study provides additional evidence on the etiologic role of drugs and vaccines in the occurrence of SJS in children.