Investigating the mechanisms underlying phytoprotection by plant growth‐promoting rhizobacteria in Spartina densiflora under metal stress

• Pollution of coasts by toxic metals and metalloids is a worldwide problem for which phytoremediation using halophytes and associated microbiomes is becoming relevant. Metal(loid) excess is a constraint for plant establishment and development, and plant growth promoting rhizobacteria (PGPR) mitigate plant stress under these conditions. However, mechanisms underlying this effect remain elusive. The effect of toxic metal(loid)s on activity and gene expression of ROS‐scavenging enzymes in roots of the halophyte Spartina densiflora grown on real polluted sediments in a greenhouse experiment was investigated.
• Sediments of the metal‐polluted joint estuary of Tinto and Odiel rivers and control, unpollutred samples from the Piedras estuary were collected and submitted to ICP‐OES. Seeds of S. densiflora were collected from the polluted Odiel marshes and grown in polluted and unpolluted sediments. Rhizophere biofilm‐forming bacteria were selected based on metal tolerance and inoculated to S. densiflora and grown for 4 months. Fresh or frozen harvested plants were used for enzyme assays and gene expression studies, respectively.
• Metal excess induced SOD (five‐fold increase), whereas CAT and ascorbate peroxidase displayed minor induction (twofold). A twofold increase of TBARs indicated membrane damage. Our results showed that metal‐resistant PGPR (P. agglomerans RSO6 and RSO7 and B. aryabhattai RSO25) contributed to alleviate metal stress, as deduced from lower levels of all antioxidant enzymes to levels below those of non‐exposed plants. The oxidative stress index (OSI) decreased between 50 and 75% upon inoculation.
• The results also evidenced the important role of PAL, involved in secondary metabolism and/or lignin synthesis, as a pathway for metal stress management in this halophyte upon inoculation with appropriate PGPR, since the different inoculation treatments enhanced PAL expression between 3.75‐ and five‐fold. Our data confirm, at the molecular level, the role of PGPR in alleviating metal stress in S. densiflora and evidence the difficulty of working with halophytes for which little genetic information is available.

     

Screening method for cDNAs encoding putative enzymes converting loganin into secologanin by a transgenic yeast culture

A screening method was developed for the detection of enzymes converting loganin to secologanin, a precursor in the biosynthesis of indole alkaloids. The method uses a transgenic yeast culture expressing two cDNAs encoding enzymes involved in the terpenoid indole alkaloid biosynthesis. In the presence of secologanin, the yeast culture produces a yellow compound visible on nitrocellulose. This color change was used to screen a cDNA library of Catharanthus roseus for a putative enzyme converting loganin into secologanin.     

Kind to kin: weak interference competition among white stork Ciconia ciconia broodmates

Altricial nestlings in structured families show a diverse array of behavioural mechanisms to compete for food, ranging from signalling scrambles to aggressive interference. Rates of filial infanticide are moderately high in white storks. It has been hypothesized that this unusual behaviour is an adaptive parental response to the absence of efficient mechanisms of brood reduction (aggression or direct physical interference) by nestlings. To test this latter assumption, we analyzed video recordings of 41 complete feeding episodes at 32 broods during the first half of the nestling period, when nestlings complete 90% of growth and chick mortality and size asymmetries are highest. Parents delivered food to all nestlings simultaneously by regurgitating on the nest floor. No direct (bill to bill) feeding was recorded. Senior nestlings were never observed to limit their junior nestlings from eating food, either by aggression or physical interference. Experimental feeding tests revealed that heavier nestlings handled prey items more efficiently and ate food at a higher speed. The high degree of tolerance shown by senior nestlings is unusual among birds with similar ecological and phylogenetic affinities, such as herons. Tolerance by seniors cannot be easily explained by absence of parental favouritism or proximate factors known to affect the occurrence of sibling aggression in other species (rate of food transfer, brood size, hatching asynchrony or length of nestling period).     

Characterization of <Emphasis Type="Italic">Penicillium</Emphasis> Species by Ribosomal DNA Sequencing and BOX,ERIC and REP-PCR Analysis

The genus Penicillium is one of the largest and widely distributed fungal genera described to date. As a result, its taxonomic classification and species discrimination within this genus has become complicated. In this study, 52 isolates that belonged to the Penicillum genus and other related genera were characterized using two DNA-based methods: (i) analysis of the nucleotide sequences of internal transcribed spacers in ribosomal DNA and (ii) analysis of DNA fingerprints that were generated by polymerase chain reactions with specific primers for enterobacterial repetitive intergenic consensus (ERIC) and repetitive extragenic palindromic (REP) sequences, and BOX elements. Using both methods, Penicillium species were discriminated from other fungal genera. Furthermore, Penicillium species that include strains which are used as biocontrol agents, such as P. glabrum, P. purpurogenum, and P. oxalicum, could be distinguished from other Penicillium species using these techniques. Based on our findings, we propose that a polyphasic approach that includes analysis of the nucleotide sequences of ribosomal DNA and detecting the presence of highly conserved, repeated nucleotide sequences can be used to determine the genetic relationships between different Penicillium species. Furthermore, we propose that our results can be used as a start point to develop a strategy to monitor the environmental presence of particular strains of Penicillium species when they are used as biocontrol agents.     

Characterization of the TRBP domain required for Dicer interaction and function in RNA interference

Background  

Dicer, Ago2 and TRBP are the minimum components of the human RNA-induced silencing complex (RISC). While Dicer and Ago2 are RNases, TRBP is the double-stranded RNA binding protein (dsRBP) that loads small interfering RNA into the RISC. TRBP binds directly to Dicer through its C-terminal domain.     

Comparison of Prognostic Gene Profiles Using qRT-PCR in Paraffin Samples: A Retrospective Study in Patients with Early Breast Cancer

Introduction

Gene profiling may improve prognostic accuracy in patients with early breast cancer, but this technology is not widely available. We used commercial assays for qRT-PCR to assess the performance of the gene profiles included in the 70-Gene Signature, the Recurrence Score and the Two-Gene Ratio.

Methods

153 patients with early breast cancer and a minimum follow-up of 5 years were included. All tumours were positive for hormonal receptors and 38% had positive lymph nodes; 64% of patients received adjuvant chemotherapy. RNA was extracted from formalin-fixed paraffin-embedded (FFPE) specimens using a specific kit. qRT-PCR amplifications were performed with TaqMan Gene Expression Assays products. We applied the three gene-expression-based models to our patient cohort to compare the predictions derived from these gene sets.

Results

After a median follow-up of 91 months, 22% of patients relapsed. The distant metastasis-free survival (DMFS) at 5 years was calculated for each profile. For the 70-Gene Signature, DMFS was 95% -good prognosis- versus 66% -poor prognosis. In the case of the Recurrence Score, DMFS was 98%, 81% and 69% for low, intermediate and high-risk groups, respectively. Finally, for the Two-Gene Ratio, DMFS was 86% versus 70%. The 70-Gene Signature and the Recurrence Score were highly informative in identifying patients with distant metastasis, even in multivariate analysis.

Conclusion

Commercially available assays for qRT-PCR can be used to assess the prognostic utility of previously published gene expression profiles in FFPE material from patients with early breast cancer. Our results, with the use of a different platform and with different material, confirm the robustness of the 70-Gene Signature and represent an independent test for the Recurrence Score, using different primer/probe sets.     

The cellular prion protein and its role in Alzheimer disease

The cellular prion protein (PrP^C) is a membrane-bound glycoprotein especially abundant in the central nervous system (CNS). The scrapie prion protein (PrP^Sc, also termed prions) is responsible of transmissible spongiform encephalopathies (TSE), a group of neurodegenerative diseases which affect humans and other mammal species, although the presence of PrP^C is needed for the establishment and further evolution of prions.The present work compares the expression and localization of PrP^C between healthy human brains and those suffering from Alzheimer disease (AD).In both situations we have observed a rostrocaudal decrease in the amount of PrP^C within the CNS, both by immunoblotting and immunohistochemistry techniques. PrP^C is higher expressed in our control brains than in AD cases. There was a neuronal loss and astogliosis in our AD cases. There was a tendency of a lesser expression of PrP^C in AD cases than in healthy ones. And in AD cases, the intensity of the expression of the unglycosylated band is higher than the di- and monoglycosylated bands.With regards to amyloid plaques, those present in AD cases were positively labeled for PrP^C, a result which is further supported by the presence of PrP^C in the amyloid plaques of a transgenic line of mice mimicking AD.The work was done according to Helsinki Declaration of 1975, and approved by the Ethics Committee of the Faculty of Medicine of the University of Navarre.Key words: cellular prion protein, Alzheimer disease, transgenic mice