Alterations in the photoactivation pathway of rhodopsin mutants associated with retinitis pigmentosa

The visual photoreceptor rhodopsin undergoes a series of conformational changes upon light activation, eventually leading to the active metarhodopsin II conformation, which is able to bind and activate the G-protein, transducin. We have previously shown that mutant rhodopsins G51V and G89D, associated with retinitis pigmentosa, present photobleaching patterns characterized by the formation of altered photointermediates whose nature remained obscure. Our current detailed UV-visible spectroscopic analysis, together with functional characterization, indicate that these mutations influence the relative stability of the different metarhodopsin photointermediates by altering their equilibria and maintaining the receptor in a nonfunctional light-induced conformation that may be toxic to photoreceptor cells. We propose that G51V and G89D shift the equilibrium from metarhodopsin I towards an intermediate, recently named as metarhodopsin Ib, proposed to interact with transducin without activating it. This may be one of the causes contributing to the molecular mechanisms underlying cell death associated with some retinitis pigmentosa mutations.     

High fragmentation characterizes tumour-derived circulating DNA

Background

Circulating DNA (ctDNA) is acknowledged as a potential diagnostic tool for various cancers including colorectal cancer, especially when considering the detection of mutations. Certainly due to lack of normalization of the experimental conditions, previous reports present many discrepancies and contradictory data on the analysis of the concentration of total ctDNA and on the proportion of tumour-derived ctDNA fragments.

Methodology

In order to rigorously analyse ctDNA, we thoroughly investigated ctDNA size distribution. We used a highly specific Q-PCR assay and athymic nude mice xenografted with SW620 or HT29 human colon cancer cells, and we correlated our results by examining plasma from metastatic CRC patients.

Conclusion/Significance

Fragmentation and concentration of tumour-derived ctDNA is positively correlated with tumour weight. CtDNA quantification by Q-PCR depends on the amplified target length and is optimal for 60–100 bp fragments. Q-PCR analysis of plasma samples from xenografted mice and cancer patients showed that tumour-derived ctDNA exhibits a specific amount profile based on ctDNA size and significant higher ctDNA fragmentation. Metastatic colorectal patients (n = 12) showed nearly 5-fold higher mean ctDNA fragmentation than healthy individuals (n = 16).     

Disease Isolates of Streptococcus pseudopneumoniae and Non-Typeable S. pneumoniae Presumptively Identified as Atypical S. pneumoniae in Spain

We aimed to obtain insights on the nature of a collection of isolates presumptively identified as atypical Streptococcus pneumoniae recovered from invasive and non-invasive infections in Spain. One-hundred and thirty-two isolates were characterized by: optochin susceptibility in ambient and CO₂-enriched atmosphere; bile solubility; PCR-based assays targeting pneumococcal genes lytA, ply, pspA, cpsA, Spn9802, aliB-like ORF2, and a specific 16S rRNA region; multilocus sequence analysis; and antimicrobial susceptibility. By multilocus sequence analysis, 61 isolates were S. pseudopneumoniae, 34 were pneumococci, 13 were S. mitis, and 24 remained unclassified as non-pneumococci. Among S. pseudopneumoniae isolates, 51 (83.6%) were collected from respiratory tract samples; eight isolates were obtained from sterile sources. High frequency of non-susceptibility to penicillin (60.7%) and erythromycin (42.6%) was found. Only 50.8% of the S. pseudopneumoniae isolates displayed the typical optochin phenotype originally described for this species. None harbored the cpsA gene or the pneumococcal typical lytA restriction fragment length polymorphism. The Spn9802 and the specific 16S rRNA regions were detected among the majority of the S. pseudopneumoniae isolates (n = 59 and n = 49, respectively). The ply and pspA genes were rarely found. A high genetic diversity was found and 59 profiles were identified. Among the S. pneumoniae, 23 were capsulated and 11 were non-typeable. Three non-typeable isolates, associated to international non-capsulated lineages, were recovered from invasive disease sources. In conclusion, half of the atypical pneumococcal clinical isolates were, in fact, S. pseudopneumoniae and one-fourth were other streptococci. We identified S. pseudopneumoniae and non-typeable pneumococci as cause of disease in Spain including invasive disease.     

Invasive Pneumococcal Disease in Healthy Adults: Increase of Empyema Associated with the Clonal-Type Sweden(1)-ST306

Background

Adult invasive pneumococcal disease (IPD) occurs mainly in the elderly and patients with co-morbidities. Little is known about the clinical characteristics, serotypes and genotypes causing IPD in healthy adults.

Methods

We studied 745 culture-proven cases of IPD in adult patients aged 18–64 years (1996–2010). Patients were included in two groups: 1.) adults with co-morbidities, and 2.) healthy adults, who had no prior or coincident diagnosis of a chronic or immunosuppressive underlying disease. Microbiological studies included pneumococcal serotyping and genotyping.

Results

Of 745 IPD episodes, 525 (70%) occurred in patients with co-morbidities and 220 (30%) in healthy adults. The healthy adults with IPD were often smokers (56%) or alcohol abusers (18%). As compared to patients with co-morbidities, the healthy adults had (P<0.05): younger age (43.5+/−13.1 vs. 48.7+/−11.3 years); higher proportions of women (45% vs. 24%), pneumonia with empyema (15% vs. 7%) and infection with non-PCV7 serotypes including serotypes 1 (25% vs. 5%), 7F (13% vs. 4%), and 5 (7% vs. 2%); and lower mortality (5% vs. 20%). Empyema was more frequently caused by serotype 1. No death occurred among 79 patients with serotype 1 IPD. There was an emergence of virulent clonal-types Sweden¹-ST306 and Netherlands^7F-ST191. The vaccine serotype coverage with the PCV13 was higher in healthy adults than in patients with co-morbidities: 82% and 56%, respectively, P<0.001.

Conclusion

In this clinical study, one-third of adults with IPD had no underlying chronic or immunosuppressive diseases (healthy adults). They were often smokers and alcohol abusers, and frequently presents with pneumonia and empyema caused by virulent clones of non-PCV7 serotypes such as the Sweden¹-ST306. Thus, implementing tobacco and alcohol abuse-cessation measures and a proper pneumococcal vaccination, such as PCV13 policy, in active smokers and alcohol abusers may diminish the burden of IPD in adults.     

Increased tolerance to Phytophthora citrophthora in transgenic orange plants constitutively expressing a tomato pathogenesis related protein PR-5

Phytophthora citrophthora is the most widely spread oomycete plant pathogen over all the citrus growing areas and represents one of the major causes of crop losses. Constitutive over-expression of genes encoding proteins involved in plant defence mechanisms to disease is one of the strategies proposed to increase plant tolerance to oomycete and fungal pathogens. P23 (PR-5), a 23-kDa pathogenesis-related protein similar to osmotins, is induced in tomato (Lycopersicon esculentum Mill. cv. Rutgers) plants when they are infected with citrus exocortis viroid, and its antifungal activity has been demonstrated in in vitro assays. We have successfully produced transgenic orange (Citrus sinensis L. Obs. cv. Pineapple) plants bearing a chimeric gene construct consisting of the cauliflower mosaic virus 35S promoter and the coding region of the tomato pathogenesis-related PR-5. Nine regenerated transgenic lines constitutively expressed the PR protein. They were challenged with Phytophthora citrophthora using a detached bark assay. A significant reduction in lesion development was consistently observed in one transgenic line in comparison to the control plants. This same line achieved plant survival rates higher than control plants when transgenic trees were inoculated with oomycete cultures. These results provide evidence for the in vivo activity of the tomato PR-5 protein against Phytophthora citrophthora, and suggest that this may be employed as a strategy aimed at engineering Phytophthora disease resistance in citrus.     

The role of E. maritimum (L.) in the dune pollination network of the Balearic Islands

Eryngium maritimum L. (Apiaceae) is a geophyte that inhabits in the dunes of the Mediterranean and Atlantic. Although it is a highly entomophilous species, there is little literature on its pollinator assemblage. The aim of this study is to analyze the role played by E. maritimum in the dune pollination network of the Balearic Islands, where there is an intense anthropogenic impact in its habitat. For this purpose, two populations located in the North and South of Mallorca were chosen, in which diurnal transects were carried out to observe and capture pollinators on 15 plant species during the anthesis period of E. maritimum. The flowering period of 10 plant species flowering at the same period than E. maritimum was analyzed to identify periods of competition. A total of 71 pollinator species were found, belonging to 30 different families. Eryngium maritimum is a strongly generalist species, with a total of 45 pollinator species. Two new species, Odice blandula and Leucospis gigas, were found for the first time in Mallorca. In terms of pollinators, Teucrium dunense and Helichrysum stoechas are the most similar species to E. maritimum. However, analysis of phenology suggests that these three species have been able to decouple their blooms to avoid competition. The present study shows that E. maritimum plays an important role in the dune pollination network, being its anthesis located at the end of the dune flowering season, when there are no functionally similar species in flower.     

Genetic diversity within<Emphasis Type="Italic"> Pisum sativum</Emphasis> using protein- and PCR-based markers

A collection of 148 Pisum accessions, mostly from Western Europe, and including both primitive germplasm and cultivated types, was structured using 121 protein- and PCR-based markers. This molecular marker-based classification allowed us to trace back major lineages of pea breeding in Western Europe over the last decades, and to follow the main breeding objectives: increase of seed weight, introduction of the afila foliage type and white flowers, and improvement of frost tolerance for winter-sown peas. The classification was largely consistent with the available pedigree data, and clearly resolved the different main varietal types according to their end-uses (fodder, food and feed peas) from exotic types and wild forms. Fodder types were further separated into two sub-groups. Feed peas, corresponding to either spring-sown or winter-sown types, were also separated, with two apparently different gene pools for winter-sown peas. The garden pea group was the most difficult to structure, probably due to a continuum in breeding of feed peas from garden types. The classification also stressed the paradox between the narrowness of the genetic basis of recent cultivars and the very large diversity available within P. sativum. A sub-collection of 43 accessions representing 96% of the whole allelic variability is proposed as a starting point for the construction of a core collection.Communicated by C. Möllers