Microsynth (Switzerland)

The chemiluminescent light-emission pathway of phenoxy-1,2-dioxetane luminophores is increasingly attracting the scientific community's attention. Dioxetane probes that undergo rapid, flash-type chemiexcitation demonstrate higher detection sensitivity than those with a slower, glow-type chemiexcitation rate. This is primarily because the rapid flash-type produces a greater number of photons within a given time. Herein, we discovered that dioxetanes fused to 7-norbornyl and homocubanyl units, present accelerated chemiexcitation rates supported by DFT computational simulations. Specifically, the 7-norbornyl and homocubanyl spirofused dioxetanes exhibited a chemiexcitation rate of 14.2-fold and 230-fold faster than the spiro-adamantyl-dioxetane, respectively. A turn-ON dioxetane probe for the detection of the enzyme β-galactosidase, containing the 7-norbornyl spirofused unit, exhibited an S/N value of 415 with low enzyme concentration. This probe demonstrated an increase in detection sensitivity towards β-galactosidase with a limit-of-detection value that is 9-fold more sensitive than that obtained by the adamantyl counterpart. Interestingly, the computed activation free energies of the homocubanyl and 7-norbornyl units were correlated with their CCsC spiro-angle, to corroborate the measured chemiexcitation rates.

We present a comprehensive analysis of the hepatic miRNA transcriptome at one month post-infection of experimental primary alveolar echinococcosis (AE), a parasitic infection caused upon ingestion of E. multilocularis eggs. Liver tissues were collected from infected and non-infected C57BL/6 mice, then small RNA libraries were prepared for next-generation sequencing (NGS). We conducted a Stem-loop RT-qPCR for validation of most dysregulated miRNAs. In infected mice, the expression levels of 28 miRNAs were significantly altered. Of these, 9 were up-regulated (fold change (FC) ≥ 1.5) and 19 were down-regulated (FC ≤ 0.66) as compared to the non-infected controls. In infected livers, mmu-miR-148a-3p and mmu-miR-101b-3p were 8- and 6-fold down-regulated, respectively, and the expression of mmu-miR-22-3p was reduced by 50%, compared to non-infected liver tissue. Conversely, significantly higher hepatic levels were noted for Mus musculus (mmu)-miR-21a-5p (FC = 2.3) and mmu-miR-122-5p (FC = 1.8). In addition, the relative mRNA expression levels of five genes (vegfa, mtor, hif1-α, fasn and acsl1) that were identified as targets of down-regulated miRNAs were significantly enhanced. All the five genes exhibited a higher expression level in livers of E. multilocularis infected mice compared to non-infected mice. Finally, we studied the issue related to functionally mature arm selection preference (5p and/or 3p) from the miRNA precursor and showed that 9 pre-miRNAs exhibited different arm selection preferences in normal versus infected liver tissues. In conclusion, this study provides first evidence that miRNAs are regulated early in primary murine AE. Our findings raise intriguing questions such as (i) how E. multilocularis affects hepatic miRNA expression;(ii) what are the alterations in miRNA expression patterns in more advanced AE-stages; and (iii) which hepatic cellular, metabolic and/or immunologic processes are modulated through altered miRNAs in AE. Thus, further research on the regulation of miRNAs during AE is needed, since miRNAs constitute an attractive potential option for development of novel therapeutic approaches against AE.

Detection of Salmonella and L. monocytogenes in food samples by current diagnostic methods requires relatively long time to results (2-6 days). Furthermore, the ability to perform environmental monitoring at the factory site for these pathogens is limited due to the need for laboratory facilities. Herein, we report new chemiluminescence probes for the ultrasensitive direct detection of viable pathogenic bacteria. The probes are composed of a bright phenoxy-dioxetane luminophore masked by triggering group, which is activated by a specific bacterial enzyme, and could detect their corresponding bacteria with an LOD value of about 600-fold lower than that of fluorescent probes. Moreover, we were able to detect a minimum of 10 Salmonella cells within 6 h incubation. The assay allows for bacterial enrichment and detection in one test tube without further sample preparation. We anticipate that this design strategy will be used to prepare analogous chemiluminescence probes for other enzymes relevant to specific bacteria detection and point-of-care diagnostics.

Premise of the study:The evolutionarily and ecologically distinct coco de mer palm Lodoicea maldivica (Arecaceae) is endemic to two islands in the Seychelles. Before colonization of the islands by man, the endangered palm formed large monodominant stands, but its natural range is now restricted to four main populations and several patches of isolated individuals. Microsatellite markers were designed to investigate the genetic structure of the remaining natural populations of L. maldivica.Methods and Results:We developed 12 polymorphic and three monomorphic microsatellite markers for this species, with a mean number of alleles per locus of 13.2 (range 5–21) and expected heterozygosity values ranging from 0.31–0.91 for the polymorphic loci.Conclusions:These markers enable us to study the patterns of genetic diversity, contemporary seed dispersal, and the fine-scale spatial genetic structure of this important conservation flagship species.

BackgroundThe mollicute Mycoplasma conjunctivae is the etiological agent leading to infectious keratoconjunctivitis (IKC) in domestic sheep and wild caprinae. Although this pathogen is relatively benign for domestic animals treated by antibiotics, it can lead wild animals to blindness and death. This is a major cause of death in the protected species in the Alps (e.g., Capra ibex, Rupicapra rupicapra).MethodsThe genome was sequenced using a combined technique of GS-FLX (454) and Sanger sequencing, and annotated by an automatic pipeline that we designed using several tools interconnected via PERL scripts. The resulting annotations are stored in a MySQL database.ResultsThe annotated sequence is deposited in the EMBL database (FM864216) and uploaded into the mollicutes database MolliGen allowing for comparative genomics.ConclusionWe show that our automatic pipeline allows for annotating a complete mycoplasma genome and present several examples of analysis in search for biological targets (e.g., pathogenic proteins).

The higher plant Arabidopsis thaliana (Arabidopsis) is an important model for identifying plant genes and determining their function. To assist biological investigations and to define chromosome structure, a coordinated effort to sequence the Arabidopsis genome was initiated in late 1996. Here we report one of the first milestones of this project, the sequence of chromosome 4. Analysis of 17.38 megabases of unique sequence, representing about 17% of the genome, reveals 3,744 protein coding genes, 81 transfer RNAs and numerous repeat elements. Heterochromatic regions surrounding the putative centromere, which has not yet been completely sequenced, are characterized by an increased frequency of a variety of repeats, new repeats, reduced recombination, lowered gene density and lowered gene expression. Roughly 60% of the predicted protein-coding genes have been functionally characterized on the basis of their homology to known genes. Many genes encode predicted proteins that are homologous to human and Caenorhabditis elegans proteins.