Transgenic Rice Plants Produced by PEG-Mediated Plasmid Uptake into Protoplasts

Embryogenic cell suspension cultures were obtained from calli developed from mature rice seeds of a Japonica type Itahan cultivar, Roncarolo. Protoplasts were isolated and transformed by PEG-treated method with plasmid pHP23 carrying the NPT Ⅱ gene which encodes resistance to antibiotic G-418. Protoplast-derived colonies were selected in presence of the inhibitor. Plants were regenerated and transplanted into soil in the green house. The presence of foreign gene in the regenerated plants was verified by PCR and Southern analysis.     

Growth Monitoring: A Survey of Current Practices of Primary Care Paediatricians in Europe

Objective

We aimed to study current practices in growth monitoring by European primary care paediatricians and to explore their perceived needs in this field.

Methods

We developed a cross-sectional, anonymous on-line survey and contacted primary care paediatricians listed in national directories in the 18 European countries with a confederation of primary care paediatricians. Paediatricians participated in the survey between April and September 2011.

Results

Of the 1,198 paediatricians from 11 European countries (response rate 13%) who participated, 29% used the 2006 World Health Organization Multicentre Growth Reference Study growth charts, 69% used national growth charts; 61% used software to draw growth charts and 79% did not use a formal algorithm to detect abnormal growth on growth charts. Among the 21% of paediatricians who used algorithms, many used non-algorithmic simple thresholds for height and weight and none used the algorithms published in the international literature. In all, 69% of paediatricians declared that a validated algorithm to monitor growth would be useful in daily practice. We found important between-country variations.

Conclusion

The varied growth-monitoring practices declared by primary care paediatricians reveals the need for standardization and evidence-based algorithms to define abnormal growth and the development of software that would use such algorithms.     

Cloning, expression, and purification of recombinant bovine rotavirus hemagglutinin, VP8*, in Escherichia coli

Rotavirus VP8* subunit is the minor trypsin cleavage product of the spike protein VP4, which is the major determinant of the viral infectivity and neutralization. To study the structure-function relationship of this fragment and to obtain type-specific reagents, substantial amounts of this protein are needed. Thus, full-length VP8* cDNA, including the entire trypsin cleavage-encoding region in gene 4, was synthesized and amplified by RT-PCR from total RNA purified from bovine rotavirus strain C486 propagated in MA104 cell culture. The extended VP8* cDNA (VP8ext) was cloned into the pGEM-T Easy plasmid and subcloned into the Escherichia coli expression plasmid pET28a(+). The correspondent 30 kDa protein was overexpressed in E. coli BL21(DE3)pLysS cells under the control of the T7 promoter. The identity and the antigenicity of VP8ext were confirmed on Western blots using anti-His and anti-rotavirus antibodies. Immobilized Ni-ion affinity chromatography was used to purify the expressed protein resulting in a yield of 4 mg of VP8ext per liter of induced E. coli culture. Our results indicate that VP8ext maintained its native antigenicity and specificity, providing a good source of antigen for the production of P type-specific immune reagents. Detailed structural analysis of pure recombinant VP8 subunit should allow a better understanding of its role in cell attachment and rotavirus tropism. Application of similar procedure to distinct rotavirus P serotypes should provide valuable P serotype-specific immune reagents for rotavirus diagnostics and epidemiologic surveys.     

Flying over water: how “On bird species diversity” influenced aquatic ecology

Habitat complexity has long been known to influence animal community structure by increasing the number of available habitats. Fifty years have passed since MacArthur brothers published the seminal paper “On bird species diversity”, which revolutionized studies of habitat structure. This paper first evidenced and quantified the relationship between species diversity (birds) and habitat structural complexity (the number of stratified layers of landscape vegetation). In this article, we aim to pay homage to R. H. MacArthur’s contribution and to briefly analyze the citation history and influence of “On bird species diversity”, focusing primarily on aquatic studies. We searched for all papers that cited “On bird species diversity” on Thomson Reuters (ISI—Web of Knowledge) and analyzed them for temporal citation trends. In addition, considering only aquatic papers, we explored whether and how habitat complexity was measured, as well as the ecological organization level, attributes of organisms, taxonomic groups and study design (observational or experimental). “On bird species diversity” citations increased over time, but this paper was less cited by limnologists compared to terrestrial and marine scientists. The majority of investigations in aquatic ecosystems quantified habitat complexity, but few used mathematical modeling. The high number of citations, which continues to increase, shows the great influence of “On bird species diversity” on ecological studies and typifies it as a classic in the ecological literature. However, the low citation frequency found in papers devoted to freshwater ecosystems indicates that limnologists in general neglect this original contribution in studies of habitat complexity.     

RAPD fingerprints for identification and for taxonomic studies of elite poplar (Populus spp.) clones

RAPD (Random Amplified Polymorphic DNA) fingerprints have recently been used to estimate genetic and taxonomic relationships in plants. In this study RAPD analysis was performed on 32 clones belonging to different species of the genus Populus. Of these, 25 clones are registered in several countries for commercial use and, altogether, cover almost 50% of the worlds cultivated poplars. DNA was prepared from leaves and amplified by PCR using random oligonucleotide primers. Amplification products were separated by agarose-gel electrophoresis to reveal band polymorphisms. Four primers out of the 18 tested, were selected on the basis of the number and frequency of the polymorphisms produced. With these a total of 120 different DNA bands were reproducibly obtained, 92% of which were polymorphic. The polymorphisms were scored and used in band-sharing analyses to identify genetic relationships. With a few but interesting exceptions, these are consistent with the present taxonomy of the genus Populus and with the known predigrees of cultivated poplars. Moreover, the results show that RAPD analysis allows one to discriminate among all tested clones and can, therefore, be recommended as a convenient tool to defend plant breeders rights.     

RNASEH1 Mutations Impair mtDNA Replication and Cause Adult-Onset Mitochondrial Encephalomyopathy

Chronic progressive external ophthalmoplegia (CPEO) is common in mitochondrial disorders and is frequently associated with multiple mtDNA deletions. The onset is typically in adulthood, and affected subjects can also present with general muscle weakness. The underlying genetic defects comprise autosomal-dominant or recessive mutations in several nuclear genes, most of which play a role in mtDNA replication. Next-generation sequencing led to the identification of compound-heterozygous RNASEH1 mutations in two singleton subjects and a homozygous mutation in four siblings. RNASEH1, encoding ribonuclease H1 (RNase H1), is an endonuclease that is present in both the nucleus and mitochondria and digests the RNA component of RNA-DNA hybrids. Unlike mitochondria, the nucleus harbors a second ribonuclease (RNase H2). All affected individuals first presented with CPEO and exercise intolerance in their twenties, and these were followed by muscle weakness, dysphagia, and spino-cerebellar signs with impaired gait coordination, dysmetria, and dysarthria. Ragged-red and cytochrome c oxidase (COX)-negative fibers, together with impaired activity of various mitochondrial respiratory chain complexes, were observed in muscle biopsies of affected subjects. Western blot analysis showed the virtual absence of RNase H1 in total lysate from mutant fibroblasts. By an in vitro assay, we demonstrated that altered RNase H1 has a reduced capability to remove the RNA from RNA-DNA hybrids, confirming their pathogenic role. Given that an increasing amount of evidence indicates the presence of RNA primers during mtDNA replication, this result might also explain the accumulation of mtDNA deletions and underscores the importance of RNase H1 for mtDNA maintenance.     

Predicting the success of an invader: Niche shift versus niche conservatism

Invasive species can encounter environments different from their source populations, which may trigger rapid adaptive changes after introduction (niche shift hypothesis). To test this hypothesis, we investigated whether postintroduction evolution is correlated with contrasting environmental conditions between the European invasive and source ranges in the Asian tiger mosquito Aedes albopictus. The comparison of environmental niches occupied in European and source population ranges revealed more than 96% overlap between invasive and source niches, supporting niche conservatism. However, we found evidence for postintroduction genetic evolution by reanalyzing a published ddRADseq genomic dataset from 90 European invasive populations using genotype–environment association (GEA) methods and generalized dissimilarity modeling (GDM). Three loci, among which a putative heat‐shock protein, exhibited significant allelic turnover along the gradient of winter precipitation that could be associated with ongoing range expansion. Wing morphometric traits weakly correlated with environmental gradients within Europe, but wing size differed between invasive and source populations located in different climatic areas. Niche similarities between source and invasive ranges might have facilitated the establishment of populations. Nonetheless, we found evidence for environmental‐induced adaptive changes after introduction. The ability to rapidly evolve observed in invasive populations (genetic shift) together with a large proportion of unfilled potential suitable areas (80%) pave the way to further spread of Ae. albopictus in Europe.     

Prospection and Evaluation of (Hemi) Cellulolytic Enzymes Using Untreated and Pretreated Biomasses in Two Argentinean Native Termites

Saccharum officinarum bagasse (common name: sugarcane bagasse) and Pennisetum purpureum (also known as Napier grass) are among the most promising feedstocks for bioethanol production in Argentina and Brazil. In this study, both biomasses were assessed before and after acid pretreatment and following hydrolysis with Nasutitermes aquilinus and Cortaritermes fulviceps termite gut digestome. The chemical composition analysis of the biomasses after diluted acid pretreatment showed that the hemicellulose fraction was partially removed. The (hemi) cellulolytic activities were evaluated in bacterial culture supernatants of termite gut homogenates grown in treated and untreated biomasses. In all cases, we detected significantly higher endoglucanase and xylanase activities using pretreated biomasses compared to untreated biomasses, carboxymethylcellulose and xylan. Several protein bands with (hemi) cellulolytic activity were detected in zymograms and two-dimensional gel electrophoresis. Some proteins of these bands or spots were identified as xylanolytic peptides by mass spectrometry. Finally, the diversity of cultured cellulolytic bacterial endosymbionts associated to both Argentinean native termite species was analyzed. This study describes, for the first time, bacterial endosymbionts and endogenous (hemi) cellulases of two Argentinean native termites as well as their potential application in degradation of lignocellulosic biomass for bioethanol production.     

Defining the importance of landscape metrics for large branchiopod biodiversity and conservation: the case of the Iberian Peninsula and Balearic Islands

The deficiency in the distributional data of invertebrate taxa is one of the major impediments acting on the bias towards the low awareness of its conservation status. The present study sets a basic framework to understand the large branchiopods distribution in the Iberian Peninsula and Balearic Islands. Since the extensive surveys performed in the late 1980s, no more studies existed updating the information for the whole studied area. The present study fills the gap, gathering together all available information on large branchiopods distribution since 1995, and analysing the effect of human population density and several landscape characteristics on their distribution, taking into consideration different spatial scales (100 m, 1 km and 10 km). In overall, 28 large branchiopod taxa (17 anostracans, 7 notostracans and 4 spinicaudatans) are known to occur in the area. Approximately 30% of the sites hosted multiple species, with a maximum of 6 species. Significant positive co-occurring species pairs were found clustered together, forming 4 different associations of large branchiopod species. In general, species clustered in the same group showed similar responses to analysed landscape characteristics, usually showing a better fit at higher spatial scales.     

Spatial Distribution of Helicobacter spp. in the Gastrointestinal Tract of Dogs

Background:  In dogs, the gastric Helicobacter spp. have been well studied, but there is little information regarding the other parts of the alimentary system. We sought to determine the spatial distribution of Helicobacter spp. in the gastrointestinal tract and the hepatobiliary system of dogs using culture-independent methods.
Materials and methods:  Samples of stomach, duodenum, ileum, cecum, colon, pancreas, liver, and bile from six dogs were evaluated for Helicobacter spp. by genus, gastric, and enterohepatic Helicobacter spp. Polymerase chain reaction, 16S rRNA gene sequence analysis, immunohistochemistry, and fluorescence in situ hybridization (FISH).
Results:  In the stomach, Helicobacter spp. DNA was detected in all six dogs, with H. bizzozeronii and H. felis identified by specific polymerase chain reaction. Helicobacter organisms were localized within the surface mucus, the lumen of gastric glands, and inside parietal cells. The small intestine harbored gastric and enterohepatic Helicobacter spp. DNA/antigen in low amounts. In the cecum and colon, Helicobacter spp. DNA, with highest similarity to H. bilis /flexispira taxon 8, H. cinaedi , and H. canis, was detected in all six dogs. Helicobacter organisms were localized at the mucosal surface and within the crypts. Gastric Helicobacter spp. DNA was detected occasionally in the large intestine, but no gastric Helicobacter spp. were present in clone libraries or detected by FISH.
Conclusions:  This study demonstrates that in addition to the stomach, the large intestine of dogs is also abundantly colonized by Helicobacter spp. Additional studies are necessary to investigate the association between enterohepatic Helicobacter spp. and presence of intestinal inflammatory or proliferative disorders in dogs.