Plastidic Phosphoglucose Isomerase Is an Important Determinant of Starch Accumulation in Mesophyll Cells, Growth, Photosynthetic Capacity, and Biosynthesis of Plastidic Cytokinins in Arabidopsis

Phosphoglucose isomerase (PGI) catalyzes the reversible isomerization of glucose-6-phosphate and fructose-6-phosphate. It is involved in glycolysis and in the regeneration of glucose-6-P molecules in the oxidative pentose phosphate pathway (OPPP). In chloroplasts of illuminated mesophyll cells PGI also connects the Calvin-Benson cycle with the starch biosynthetic pathway. In this work we isolated pgi1-3, a mutant totally lacking pPGI activity as a consequence of aberrant intron splicing of the pPGI encoding gene, PGI1. Starch content in pgi1-3 source leaves was ca. 10-15% of that of wild type (WT) leaves, which was similar to that of leaves of pgi1-2, a T-DNA insertion pPGI null mutant. Starch deficiency of pgi1 leaves could be reverted by the introduction of a sex1 null mutation impeding beta-amylolytic starch breakdown. Although previous studies showed that starch granules of pgi1-2 leaves are restricted to both bundle sheath cells adjacent to the mesophyll and stomata guard cells, microscopy analyses carried out in this work revealed the presence of starch granules in the chloroplasts of pgi1-2 and pgi1-3 mesophyll cells. RT-PCR analyses showed high expression levels of plastidic and extra-plastidic beta-amylase encoding genes in pgi1 leaves, which was accompanied by increased beta-amylase activity. Both pgi1-2 and pgi1-3 mutants displayed slow growth and reduced photosynthetic capacity phenotypes even under continuous light conditions. Metabolic analyses revealed that the adenylate energy charge and the NAD(P) H/NAD(P) ratios in pgi1 leaves were lower than those of WT leaves. These analyses also revealed that the content of plastidic 2-C-methyl-D-erythritol 4-phosphate (MEP)-pathway derived cytokinins (CKs) in pgi1 leaves were exceedingly lower than in WT leaves. Noteworthy, exogenous application of CKs largely reverted the low starch content phenotype of pgi1 leaves. The overall data show that pPGI is an important determinant of photosynthesis, energy status, growth and starch accumulation in mesophyll cells likely as a consequence of its involvement in the production of OPPP/glycolysis intermediates necessary for the synthesis of plastidic MEP-pathway derived hormones such as CKs.

Genotypes, Recombinant Forms, and Variants of Norovirus GII.4 in Gipuzkoa (Basque Country, Spain), 2009-2012

Background: Noroviruses (NoVs) are genetically diverse, with genogroup II-and within it-genotype 4 (GII.4) being the most prevalent cause of acute gastroenteritis worldwide. The aim of this study was to characterize genogroup II NoV causing acute gastroenteritis in the Basque Country (northern Spain) from 2009-2012. Methods: The presence of NoV RNA was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) in stool specimens from children younger than 15 years old with community-acquired acute gastroenteritis, and from hospitalized adults or elderly residents of nursing homes with acute gastroenteritis. For genotyping, the open reading frames ORF1 (encoding the polymerase) and ORF2 (encoding the major capsid protein) were partially amplified and sequenced. Recombinant strains were confirmed by PCR of the ORF1/ORF2 junction region. Results: NoV was detected in 16.0% (453/2826) of acute gastroenteritis episodes in children younger than 2 years, 9.9% (139/1407) in children from 2 to 14 years, and 35.8% (122/341) in adults. Of 317 NoVs characterized, 313 were genogroup II and four were genogroup I. The GII.4 variants Den Haag-2006b and New Orleans-2009 predominated in 2009 and 2010-2011, respectively. In 2012, the New Orleans-2009 variant was partially replaced by the Sydney-2012 variant (GII.Pe/GII.4) and New Orleans-2009/Sydney-2012 recombinant strains. The predominant capsid genotype in all age groups was GII.4, which was the only genotype detected in outbreaks. The second most frequent genotype was GII.3 (including the recently described recombination GII.P16/GII.3), which was detected almost exclusively in children. Conclusion: Nine different genotypes of NoV genogroup II were detected; among these, intergenotype recombinant strains represented an important part, highlighting the role of recombination in the evolution of NoVs. Detection of new NoV strains, not only GII.4 strains, shortly after their first detection in other parts of the world shows that many NoV strains can spread rapidly.

Dipeptidyl-Peptidase IV Activity Is Correlated with Colorectal Cancer Prognosis

Background Dipeptidyl-peptidase IV (EC 3.4.14.5) (DPPIV) is a serine peptidase involved in cell differentiation, adhesion, immune modulation and apoptosis, functions that control neoplastic transformation. Previous studies have demonstrated altered expression and activity of tissue and circulating DPPIV in several cancers and proposed its potential usefulness for early diagnosis in colorectal cancer (CRC). Methods and principal findings The activity and mRNA and protein expression of DPPIV was prospectively analyzed in adenocarcinomas, adenomas, uninvolved colorectal mucosa and plasma from 116 CRC patients by fluorimetric, quantitative RT-PCR and immunohistochemical methods. Results were correlated with the most important classic pathological data related to aggressiveness and with 5-year survival rates. Results showed that: 1) mRNA levels and activity of DPPIV increased in colorectal neoplasms (Kruskal-Wallis test, p<0.01); 2) Both adenomas and CRCs displayed positive cytoplasmic immunostaining with luminal membrane reinforcement; 3) Plasmatic DPPIV activity was lower in CRC patients than in healthy subjects (Mann-U test, p<0.01); 4) Plasmatic DPPIV activity was associated with worse overall and disease-free survivals (log-rank p<0.01, Cox analysis p<0.01). Conclusion/significance 1) Up-regulation of DPPIV in colorectal tumors suggests a role for this enzyme in the neoplastic transformation of colorectal tissues. This finding opens the possibility for new therapeutic targets in these patients. 2) Plasmatic DPPIV is an independent prognostic factor in survival of CRC patients. The determination of DPPIV activity levels in the plasma may be a safe, minimally invasive and inexpensive way to define the aggressiveness of CRC in daily practice.

18S rRNA V9 metabarcoding for diet characterization: a critical evaluation with two sympatric zooplanktivorous fish species

The potential of the 18S rRNA V9 metabarcoding approach for diet assessment was explored using MiSeq paired-end (PE; 2 9 150 bp) technology. To critically evaluate the method's performance with degraded/digested DNA, the diets of two zooplanktivorous fish species from the Bay of Biscay, European sardine (Sardina pilchardus) and European sprat (Sprattus sprattus), were analysed. The taxonomic resolution and quantitative potential of the 18S V9 metabarcoding was first assessed both in silico and with mock and field plankton samples. Our method was capable of discriminating species within the reference database in a reliable way providing there was at least one variable position in the 18S V9 region. Furthermore, it successfully discriminated diet between both fish species, including habitat and diel differences among sardines, overcoming some of the limitations of traditional visual-based diet analysis methods. The high sensitivity and semi-quantitative nature of the 18S V9 metabarcoding approach was supported by both visual microscopy and qPCR-based results. This molecular approach provides an alternative cost and time effective tool for food-web analysis.