Real-Time PCR Assays for the Specific Identification of Probiotic Strains Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242)

The broad spectrum of health benefits attributed to probiotics has contributed to a rapid increase in the value of the probiotic market. Probiotic health benefits can be strain specific. Thus, strain-level identification of probiotic strains is of paramount importance to ensure probiotic efficacy. Both Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC (NCIMB 30242) strains have clinically proven health benefits; however, no assays were developed to enable strain-level identification of either of these strains. The objective of this study is to develop strain-specific PCR-based methods for Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains, and to validate these assays according to the guidelines for validating qualitative real-time PCR assays. Using RAST (Rapid Annotation using Subsystem Technology), unique sequence regions were identified in the genome sequences of both strains. Probe-based assays were designed and validated for specificity, sensitivity, efficiency, repeatability, and reproducibility. Both assays were specific to target strain with 100% true positive and 0% false positive rates. Reaction efficiency for both assays was in the range of 90 to 108% with R square values > 0.99. Repeatability and reproducibility were evaluated using five samples at three DNA concentrations each and relative standard deviation was < 4% for repeatability and < 8% for reproducibility. Both of the assays developed and validated in this study for the specific identification of Lactobacillus gasseri BNR17 and Lactobacillus reuteri LRC strains are specific, sensitive, and precise. These assays can be applied to evaluate and ensure compliance in probiotic products.

     

A benign approach to the preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM) imaging

Abstract

Several different species of freshwater Bryozoa, belonging to the genera Plumatella, Rumarcanella and Fredericella, were detected within the Northern Mallee Pipeline (NMP) system in Victoria, Australia, that required definitive identification. These organisms produce asexual buds called statoblasts, with valves composed of sclerotised chitin that bear minute micro-ornamentations of considerable taxonomical significance. Imaging and analysis of these distinctive micro-ornamentations using scanning electron microscopy (SEM) is often employed for species identification. Meticulous preparation of statoblast samples is therefore required that necessitates the removal of adhering debris, dehydration and drying—whilst mitigating specimen damage and distortion. This technical note describes an approach whereby each of these three steps have been individually designed to be as benign as possible, using mild detergent/sonication to remove debris, a gradual and gentle dehydration procedure using ethanol, and critical point drying. For the overall process, these methods are chosen to optimise control and to minimise the use of harsh and hazardous chemicals.     

The use of adjunctive GPIIb/IIIa inhibitors in patients with unstable angina/non-Q-wave MI undergoing percutaneous coronary intervention

Glycoprotein IIb/IIIa receptor inhibitors represent a relatively new therapeutic approach in the field of antiplatelet therapy. Following the development of abciximab a number of small molecule GPIIb/IIIa inhibitors have been introduced such as tirofiban and eptifibatide. In this fast-moving field the interventional cardiologist needs a framework to guide decision-making for the individual patient. This review covers the efficacy and safety data from the clinical trials of GPIIb/IIIa inhibitors in the context of patients undergoing percutaneous coronary intervention for unstable angina/non-Q-wave myocardial infarction. There is an increasing body of evidence to support the efficacy of GPIIb/IIIa inhibitors in reducing the risk of adverse ischemic events in high and low risk patients undergoing percutaneous coronary intervention. A number of unresolved efficacy and safety issues remain, including the duration of treatment before and after intervention; whether a reduction in the heparin dose would further decrease the risk of hemorrhage without affecting the periprocedural thrombotic rate in patients undergoing PTCA with adjunctive GPIIb/IIIa inhibitors; and the cost-effectiveness of this therapy. When a thorough analysis of cost-effectiveness has been made, it will be easier to advocate the widespread use of these agents in all patients undergoing coronary intervention.     

A high-throughput cloning system for reverse genetics in <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>

Background  

The three trypanosomatids pathogenic to men, Trypanosoma cruzi, Trypanosoma brucei and Leishmania major, are etiological agents of Chagas disease, African sleeping sickness and cutaneous leishmaniasis, respectively. The complete sequencing of these trypanosomatid genomes represented a breakthrough in the understanding of these organisms. Genome sequencing is a step towards solving the parasite biology puzzle, as there are a high percentage of genes encoding proteins without functional annotation. Also, technical limitations in protein expression in heterologous systems reinforce the evident need for the development of a high-throughput reverse genetics platform. Ideally, such platform would lead to efficient cloning and compatibility with various approaches. Thus, we aimed to construct a highly efficient cloning platform compatible with plasmid vectors that are suitable for various approaches.     

Spatial patterns of plant diversity below-ground as revealed by DNA barcoding

Our understanding of the spatial organization of root diversity in plant communities and of the mechanisms of community assembly has been limited by our ability to identify plants based on root tissue, especially in diverse communities. Here, we test the effectiveness of the plastid gene rbcL, a core plant DNA barcoding marker, for investigating spatial patterns of root diversity, and relate observed patterns to above-ground community structure. We collected 3800 root fragments from four randomly positioned, 1-m-deep soil profiles (two vertical transects per plot), located in an old-field community in southern Ontario, Canada, and extracted and sequenced DNA from 1531 subsampled fragments. We identified species by comparing sequences with a DNA barcode reference library developed previously for the local flora. Nearly 85% of sampled root fragments were successfully sequenced and identified as belonging to 29 plant species or species groups. Root abundance and species richness varied in horizontal space and were negatively correlated with soil depth. The relative abundance of taxa below-ground was correlated with their frequency above-ground (r = 0.73, P = 0.0001), but several species detected in root tissue were not observed in above-ground quadrats. Multivariate analyses indicated that diversity was highly structured below-ground, and associated with depth, root morphology, soil chemistry and soil texture, whereas little structure was evident above-ground. Furthermore, analyses of species co-occurrence indicates strong species segregation overall but random co-occurrence among confamilials. Our results provide insights into the role of environmental filtering and competitive interactions in the organization of plant diversity below-ground, and also demonstrate the utility of barcoding for the identification of plant roots.     

Testing candidate plant barcode regions in the Myristicaceae

The concept and practice of DNA barcoding have been designed as a system to facilitate species identification and recognition. The primary challenge for barcoding plants has been to identify a suitable region on which to focus the effort. The slow relative nucleotide substitution rates of plant mitochondria and the technical issues with the use of nuclear regions have focused attention on several proposed regions in the plastid genome. One of the challenges for barcoding is to discriminate closely related or recently evolved species. The Myristicaceae, or nutmeg family, is an older group within the angiosperms that contains some recently evolved species providing a challenging test for barcoding plants. The goal of this study is to determine the relative utility of six coding (Universal Plastid Amplicon - UPA, rpoB, rpoc1, accD, rbcL, matK) and one noncoding (trnH-psbA) chloroplast loci for barcoding in the genus Compsoneura using both single region and multiregion approaches. Five of the regions we tested were predominantly invariant across species (UPA, rpoB, rpoC1, accD, rbcL). Two of the regions (matK and trnH-psbA) had significant variation and show promise for barcoding in nutmegs. We demonstrate that a two-gene approach utilizing a moderately variable region (matK) and a more variable region (trnH-psbA) provides resolution among all the Compsonuera species we sampled including the recently evolved C. sprucei and C. mexicana. Our classification analyses based on nonmetric multidimensional scaling ordination, suggest that the use of two regions results in a decreased range of intraspecific variation relative to the distribution of interspecific divergence with 95% of the samples correctly identified in a sequence identification analysis.     

Immunoprophylaxis of respiratory syncytial virus: global experience

In sarcoidosis, host genetic factors are discussed as contributing to disease susceptibility and course. Since tumor necrosis factor (TNF)-α is a central mediator of granuloma formation and since elevated TNF-α levels are found during active phases of sarcoidosis, genetic polymorphisms correlating with influences on TNF-α levels are of special interest. The complete sequencing of the MHC region and the increase in the number of identified gene polymorphisms in this locus associated with TNF-α production offer the opportunity of detecting new genes associated with sarcoidosis and perhaps of defining disease-associated haplotypes that bear the potential of serving as predictive markers for this disease.     

Flexibility in crosstalk between H2B ubiquitination and H3 methylation in vivo

Histone H2B ubiquitination is a dynamic modification that promotes methylation of histone H3K79 and H3K4. This crosstalk is important for the DNA damage response and has been implicated in cancer. Here, we show that in engineered yeast strains, ubiquitins tethered to every nucleosome promote H3K79 and H3K4 methylation from a proximal as well as a more distal site, but only if in a correct orientation. This plasticity indicates that the exact location of the attachment site, the native ubiquitin-lysine linkage and ubiquitination cycles are not critical for trans-histone crosstalk in vivo. The flexibility in crosstalk also indicates that other ubiquitination events may promote H3 methylation.