Secondary structure prediction and in vitro accessibility of mRNA as tools in the selection of target sites for ribozymes

We have investigated the relative merits of two commonly used methods for target site selection for ribozymes: secondary structure prediction (MFold program) and in vitro accessibility assays. A total of eight methylated ribozymes with DNA arms were synthesized and analyzed in a transient co-transfection assay in HeLa cells. Residual expression levels ranging from 23 to 72% were obtained with anti-PSKH1 ribozymes compared to cells transfected with an irrelevant control ribozyme. Ribozyme efficacy depended on both ribozyme concentration and the steady state expression levels of the target mRNA. Allylated ribozymes against a subset of the target sites generally displayed poorer efficacy than their methylated counterparts. This effect appeared to be influenced by in vivo accessibility of the target site. Ribozymes designed on the basis of either selection method displayed a wide range of efficacies with no significant differences in the average activities of the two groups of ribozymes. While in vitro accessibility assays had limited predictive power, there was a significant correlation between certain features of the predicted secondary structure of the target sequence and the efficacy of the corresponding ribozyme. Specifically, ribozyme efficacy appeared to be positively correlated with the presence of short stem regions and helices of low stability within their target sequences. There were no correlations with predicted free energy or loop length.     

Primers and polymerase chain reaction conditions for DNA barcoding teleost fish based on the mitochondrial cytochrome b and nuclear rhodopsin genes

This report describes a set of 21 polymerase chain reaction primers and amplification conditions developed to barcode practically any teleost fish species according to their mitochondrial cytochrome b and nuclear rhodopsin gene sequences. The method was successfully tested in more than 200 marine fish species comprising the main Actinopterygii family groups. When used in phylogenetic analyses, its combination of two genes with different evolutionary rates serves to identify fish at the species level. We provide a flow diagram indicating our validated polymerase chain reaction amplification conditions for barcoding and species identification applications as well as population structure or haplotyping analyses, adaptable to high‐throughput analyses.     

Human Breast Milk miRNA,Maternal Probiotic Supplementation and Atopic Dermatitis in Offspring

Background

Perinatal probiotic ingestion has been shown to prevent atopic dermatitis (AD) in infancy in a number of randomised trials. The Probiotics in the Prevention of Allergy among Children in Trondheim (ProPACT) trial involved a probiotic supplementation regime given solely to mothers in the perinatal period and demonstrated a ~40% relative risk reduction in the cumulative incidence of AD at 2 years of age. However, the mechanisms behind this effect are incompletely understood. Micro-RNAs (miRNA) are abundant in mammalian milk and may influence the developing gastrointestinal and immune systems of newborn infants. The objectives of this study were to describe the miRNA profile of human breast milk, and to investigate breast milk miRNAs as possible mediators of the observed preventative effect of probiotics.

Methods

Small RNA sequencing was conducted on samples collected 3 months postpartum from 54 women participating in the ProPACT trial. Differential expression of miRNA was assessed for the probiotic vs placebo and AD vs non-AD groups. The results were further analysed using functional prediction techniques.

Results

Human breast milk samples contain a relatively stable core group of highly expressed miRNAs, including miR-148a-3p, miR-22-3p, miR-30d-5p, let-7b-5p and miR-200a-3p. Functional analysis of these miRNAs revealed enrichment in a broad range of biological processes and molecular functions. Although several miRNAs were found to be differentially expressed on comparison of the probiotic vs placebo and AD vs non-AD groups, none had an acceptable false discovery rate and their biological significance in the development of AD is not immediately apparent from their predicted functional consequences.

Conclusion

Whilst breast milk miRNAs have the potential to be active in a diverse range of tissues and biological process, individual miRNAs in breast milk 3 months postpartum are unlikely to play a major role in the prevention of atopic dermatitis in infancy by probiotics ingestion in the perinatal period.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00159523","term_id":"NCT00159523"}}NCT00159523     

Examining the effects of species richness on community stability: an assembly model approach

We build dynamic models of community assembly by starting with one species in our model ecosystem and adding colonists. We find that the number of species present first increases, then fluctuates about some level. We ask: how large are these fluctuations and how can we characterize them statistically? As in Robert May's work, communities with weaker interspecific interactions permit a greater number of species to coexist on average. We find that as this average increases, however, the relative variation in the number of species and return times to mean community levels decreases. In addition, the relative frequency of large extinction events to small extinction events decreases as mean community size increases. While the model reproduces several of May's results, it also provides theoretical support for Charles Elton's idea that diverse communities such as those found in the tropics should be less variable than depauperate communities such as those found in arctic or agricultural settings.     

Assessing the reliability of eBURST using simulated populations with known ancestry

Background  

The program eBURST uses multilocus sequence typing data to divide bacterial populations into groups of closely related strains (clonal complexes), predicts the founding genotype of each group, and displays the patterns of recent evolutionary descent of all other strains in the group from the founder. The reliability of eBURST was evaluated using populations simulated with different levels of recombination in which the ancestry of all strains was known.     

Functional changes in human leukemic cell line HL-60. A model for myeloid differentiation

Polar solvents induce terminal differentiation in the human promyelocytic leukemia cell line HL-60. The present studies describe the functional changes that accompany the morphologic progression from promyelocytes to bands and poly-morphonuclear leukocytes (PMN) over 9 d of culture in 1.3 percent dimethylsulfoxide (DMSO). As the HL-60 cells mature, the rate of O(2-) production increase 18-fold, with a progressive shortening of the lag time required for activation. Hexosemonophosphate shunt activity rises concomitantly. Ingestin of paraffin oil droplets opsonized with complement or Ig increases 10-fold over 9 d in DMSO. Latex ingestion per cell by each morphologic type does not change significantly, but total latex ingestion by groups of cells increases with the rise in the proportion of mature cells with greater ingestion capacities. Degranulation, as measured by release of β-glucuronidase, lysozyme, and peroxidase, reaches maximum after 3-6 d in DMSO, then declines. HL-60 cells contain no detectable lactoferrin, suggesting that their secondary granules are absent or defective. However, they kill staphylococci by day 6 in DMSO. Morphologically immature cells (days 1-3 in DMSO) are capable of O(2-) generation, hexosemonophosphate shunt activity, ingestion, degranulation, and bacterial killing. Maximal performance of each function by cells incubated in DMSO for longer periods of time is 50-100 percent that of normal PMN. DMSO- induced differentiation of HL-60 cells is a promising model for myeloid development.     

PheMaDB: A solution for storage,retrieval, and analysis of high throughput phenotype data

Background  

OmniLog™ phenotype microarrays (PMs) have the capability to measure and compare the growth responses of biological samples upon exposure to hundreds of growth conditions such as different metabolites and antibiotics over a time course of hours to days. In order to manage the large amount of data produced from the OmniLog™ instrument, PheMaDB (Phenotype Microarray DataBase), a web-based relational database, was designed. PheMaDB enables efficient storage, retrieval and rapid analysis of the OmniLog™ PM data.     

Contrasting population structures in two sympatric anurans: implications for species conservation

A general prediction of the neutral theory of evolution is that genetic diversity should correlate positively with effective population size. We show here that diversity across eight microsatellite loci was consistently and substantially lower in one common amphibian (Bufo bufo) than in another with similar life history traits (Rana temporaria) despite B. bufo having the larger breeding assemblage sizes. However, B. bufo breeding assemblages were much more highly differentiated than those of R. temporaria according to both Fst and Rst estimators. These differences occurred in shared habitats across identical geographical distances. The patterns of genetic diversity and differentiation detected in these two species were probably a consequence of high gene flow in R. temporaria but much lower gene flow among the larger but more dispersed B. bufo assemblages. These observations highlight the difficulty of defining the boundaries of wild populations, and show how two broadly similar species can exhibit very different population dynamics.     

Isolation and cross‐familial amplification of 41 microsatellites for the brook charr (Salvelinus fontinalis)

The brook charr (Salvelinus fontinalis; Osteichthyes: Salmonidae) is a phenotypically diverse fish species inhabiting much of North America. But relatively few genetic diagnostic resources are available for this fish species. We isolated 41 microsatellites from S. fontinalis polymorphic in one or more species of salmonid fish. Thirty‐seven were polymorphic in brook charr, 15 in the congener Arctic charr (Salvelinus alpinus) and 14 in the lake charr (Salvelinus namaycush). Polymorphism was also relatively high in Oncorhynchus, where 21 loci were polymorphic in rainbow trout (Oncorhynchus mykiss) and 16 in cutthroat trout (Oncorhynchus clarkii) but only seven and four microsatellite loci were polymorphic in the more distantly related lake whitefish (Coregonus clupeaformis) and Atlantic salmon (Salmo salar), respectively. One duplicated locus (Sfo228Lav) was polymorphic at both duplicates in S. fontinalis.