Early Changes in Skeletal Muscle of Young C22 Mice, A Model of Charcot-Marie-Tooth 1A

Background: The C22 mouse is a Charcot-Marie-Tooth 1A transgenic model with minimal axonal loss. Objective: To analyse early skeletal muscle changes resulting from this dysmyelinating neuropathy. Methods: Histology of tibialis anterior muscles of C22 mice and wild type litter mate controls for morphometric analysis and (immuno-)histochemistry for known denervation markers and candidate proteins identified by representational difference analysis (RDA) based on mRNA from the same muscles; quantitative PCR and Western blotting for confirmation of RDA findings. Results: At age 10 days, morphometry was not different between groups, while at 21 days, C22 showed significantly more small diameter fibres, indicating the onset of atrophy at an age when weakness becomes detectable. Neither (immuno-)histochemistry nor RDA detected extrajunctional expression of acetylcholine receptors by age 10 and 21 days, respectively. RDA identified some mRNA up-regulated in C22 muscles, among them at 10 days, prior to detectable weakness or atrophy, integral membrane protein 2a (Itm2a), eukaryotic initiation factor 2, subunit 2 (Eif2s2) and cytoplasmic phosphatidylinositol transfer protein 1 (Pitpnc1). However, qPCR failed to measure significant differences. In contrast, Itm2a and Eif2s2 mRNA were significantly down-regulated comparing 21 versus 10 days of age in both groups, C22 and controls. Western blotting confirmed significant down-regulation of ITM2A protein in C22 only. Conclusion: Denervation-like changes in this model develop slowly with onset of atrophy and weakness at about three weeks of age, before detection of extrajunctional acetylcholine receptors. Altered Itm2a expression seems to being early as an increase, but becomes distinct as a decrease later.

Representational Difference Analysis of Transcripts Involved in Jervine Biosynthesis

Veratrum-type steroidal alkaloids (VSA) are the major bioactive ingredients that strongly determine the pharmacological activities of Veratrum nigrum. Biosynthesis of VSA at the molecular and genetic levels is not well understood. Next-generation sequencing of representational difference analysis (RDA) products after elicitation and precursor feeding was applied to identify candidate genes involved in VSA biosynthesis. A total of 12,048 contigs with a median length of 280 bases were received in three RDA libraries obtained after application of methyl jasmonate, squalene and cholesterol. The comparative analysis of annotated sequences was effective in identifying candidate genes. GABAT2 transaminase and hydroxylases active at C-22, C-26, C-11, and C-16 positions in late stages of jervine biosynthesis were selected. Moreover, genes coding pyrroline-5-carboxylate reductase and enzymes from the short-chain dehydrogenases/reductases family (SDR) associated with the reduction reactions of the VSA biosynthesis process were proposed. The data collected contribute to better understanding of jervine biosynthesis and may accelerate implementation of biotechnological methods of VSA biosynthesis.

Factors promoting the natural regeneration of Larix principis-rupprechtii plantation in the Lvliang Mountains of central China

Given their complexity, targeted care and management of different areas and tree species are necessary for enhancing the natural regeneration of forests. Thus, an understanding of changes in the overstory and understory is essential for ensuring successful regeneration. Promoting the natural regeneration of Larix principis-rupprechtii plantations is considered challenging; indeed, regional sustainable development through natural regeneration of many stands has often been considered unattainable. Here, we studied several plots with varying extents of regeneration to identify the most important factors that affect regeneration. The plots were divided into three forest types based on the number of regenerating plants. For each type of plot, we measured various factors that might potentially affect regeneration. Representational difference analysis was used to identify the most important factors >9% contribution). Based on these factors, multiple corrections were made to construct a structural equation model of topography, stand structure, soil properties and litter to identify the most important factors driving variation in regeneration. Positive correlations were detected between regeneration with diameter at breast height (0.21) and litter thickness (0.57). Regeneration was negatively correlated with soil (−0.54) and slope (−0.48). Additionally, the number of regenerating plants and the height of regenerating plants were strongly positively correlated. However, there was no significant relationship between regeneration and litter accumulation, stand density, altitude, average tree height, total P and total N. Overall, our study showed that key factors for promoting natural regeneration include appropriate litter thickness, strong parent trees, a gentle slope and sufficient quantities of soil nutrients. Moreover, our findings provide a reference for the design of effective management and restoration plans.

Strategies to develop strain-specific PCR based assays for probiotics

Since health benefits conferred by probiotics are strain-specific, identification to the strain level is mandatory to allow the monitoring of the presence and the abundance of specific probiotic in a product or in a gastrointestinal tract. Compared to standard plate counts, the reduced duration of the assays and higher specificity makes PCR-based methods (standard PCR and quantitative PCR) very appropriate for detection or quantification of probiotics. Development of strain-specific assay consists of 4 main stages: (1) strain-specific marker identification; (2) construction of potential strain-specific primers; (3) validation on DNA from pure cultures of target and related strains; and (4) validation on spiked samples. The most important and also the most challenging step is the identification of strain-specific sequences, which can be subsequently targeted by specific primers or probes. Such regions can be identified on sequences derived from 16S-23S internally transcribed spacers, randomly amplified polymorphic DNA, representational difference analysis and suppression subtractive hybridisation. Already known phenotypic or genotypic characteristics of the target strain can also be used to develop the strain-specific assay. However, the initial stage of strain-specific assay development can be replaced by comparative genomics analysis of target genome with related genomes in public databases. Advances in whole genome sequencing (WGS) have resulted in a cost reduction for bacterial genome sequencing and consequently have made this approach available to most laboratories. In the present paper I reviewed the available literature on PCR and qPCR assays developed for detection of a specific probiotic strain and discussed future WGS and comparative genomics-based approaches.

Development and application of molecular biomarkers for characterizing Caribbean Yellow Band Disease inOrbicella faveolata

Molecular stress responses associated with coral diseases represent an under-studied area of cnidarian transcriptome investigations. Caribbean Yellow Band Disease (CYBD) is considered a disease ofSymbiodiniumwithin the tissues of the coral hostOrbicella faveolata. There is a paucity of diagnostic tools to assist in the early detection and characterization of coral diseases. The validity of a diagnostic test is determined by its ability to distinguish host organisms that have the disease from those that do not. The ability to detect and identify disease-affected tissue before visible signs of the disease are evident would then be a useful diagnostic tool for monitoring and managing disease outbreaks. Representational Difference Analysis (RDA) was utilized to isolate differentially expressed genes inO. faveolataexhibiting CYBD. Preliminary screening of RDA products identified a small number of genes of interest (GOI) which included an early growth response factor and ubiquitin ligase from the coral host as well as cytochrome oxidase from the algal symbiont. To further characterize the specificity of response, quantitative real-time PCR (qPCR) was utilized to compare the expression profiles of these GOIs within diseased tissues (visible lesions), tissues that precede visible lesions by 2–4 cm (transition area), and tissues from healthy-looking colonies with no signs of disease. Results show there are distinctive differences in the expression profiles of these three GOIs within each tissue examined. Collectively, this small suite of GOIs can provide a molecular “finger print” which is capable of differentiating between infected and uninfected colonies on reefs where CYBD is known to occur.