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.

     

SSR-based linkage map of flax (<Emphasis Type="Italic">Linum usitatissimum</Emphasis> L.) and mapping of QTLs underlying fatty acid composition traits

Flax (Linum usitatissimum L.) seeds contain nearly 50% oil which is high in linolenic acid (an omega-3 fatty acid). In this study, a genetic linkage map was constructed based on 114 expressed sequence tag-derived simple sequence repeat (SSR) markers in addition to five single nucleotide polymorphism markers, five genes (fad2A, fad2B, fad3A, fad3B and dgat1) and one phenotypic trait (seed coat color), using a doubled haploid (DH) population of 78 individuals generated from a cross between SP2047 (a yellow-seeded Solin™ line with 2–4% linolenic acid) and UGG5-5 (a brown-seeded flax line with 63–66% linolenic acid). This map consists of 24 linkage groups with 113 markers spanning ~833.8 cM. Quantitative trait locus (QTL) analysis detected two major QTLs each for linoleic acid (LIO, QLio.crc-LG7, QLio.crc-LG16), linolenic acid (LIN, QLin.crc-LG7, QLin.crc-LG16) and iodine value (IOD, QIod.crc-LG7, QIod.crc-LG16), and one major QTL for palmitic acid (PAL, QPal.crc-LG9). The mutant allele of fad3A, mapped to the chromosomal segment inherited from the parent SP2047, underlies the QTL on linkage group 7 and was positively associated with high LIO content but negatively associated with LIN and IOD. This fad3A locus accounted for approximately 34, 25 and 29% of the phenotypic variation observed in this DH population for these three traits, respectively. The QTL localized on linkage group 16 explained approximately 20, 25 and 13% of the phenotypic variation for these same traits, respectively. For palmitic acid, QPal.crc-LG9 accounted for ~42% of the phenotypic variation. This first SSR-based linkage map in flax will serve as a resource for mapping additional markers, genes and traits, in map-based cloning and in marker-assisted selection.     

Integrated consensus genetic and physical maps of flax (Linum usitatissimum L.)

Three linkage maps of flax (Linum usitatissimum L.) were constructed from populations CDC Bethune/Macbeth, E1747/Viking and SP2047/UGG5-5 containing between 385 and 469 mapped markers each. The first consensus map of flax was constructed incorporating 770 markers based on 371 shared markers including 114 that were shared by all three populations and 257 shared between any two populations. The 15 linkage group map corresponds to the haploid number of chromosomes of this species. The marker order of the consensus map was largely collinear in all three individual maps but a few local inversions and marker rearrangements spanning short intervals were observed. Segregation distortion was present in all linkage groups which contained 1–52 markers displaying non-Mendelian segregation. The total length of the consensus genetic map is 1,551?cM with a mean marker density of 2.0?cM. A total of 670 markers were anchored to 204 of the 416 fingerprinted contigs of the physical map corresponding to ~274?Mb or 74?% of the estimated flax genome size of 370?Mb. This high resolution consensus map will be a resource for comparative genomics, genome organization, evolution studies and anchoring of the whole genome shotgun sequence.     

Local Knowledge and Conservation of Seagrasses in the Tamil Nadu State of India

Local knowledge systems are not considered in the conservation of fragile seagrass marine ecosystems. In fact, little is known about the utility of seagrasses in local coastal communities. This is intriguing given that some local communities rely on seagrasses to sustain their livelihoods and have relocated their villages to areas with a rich diversity and abundance of seagrasses. The purpose of this study is to assist in conservation efforts regarding seagrasses through identifying Traditional Ecological Knowledge (TEK) from local knowledge systems of seagrasses from 40 coastal communities along the eastern coast of India. We explore the assemblage of scientific and local traditional knowledge concerning the 1. classification of seagrasses (comparing scientific and traditional classification systems), 2. utility of seagrasses, 3. Traditional Ecological Knowledge (TEK) of seagrasses, and 4. current conservation efforts for seagrass ecosystems. Our results indicate that local knowledge systems consist of a complex classification of seagrass diversity that considers the role of seagrasses in the marine ecosystem. This fine-scaled ethno-classification gives rise to five times the number of taxa (10 species = 50 local ethnotaxa), each with a unique role in the ecosystem and utility within coastal communities, including the use of seagrasses for medicine (e.g., treatment of heart conditions, seasickness, etc.), food (nutritious seeds), fertilizer (nutrient rich biomass) and livestock feed (goats and sheep). Local communities are concerned about the loss of seagrass diversity and have considerable local knowledge that is valuable for conservation and restoration plans. This study serves as a case study example of the depth and breadth of local knowledge systems for a particular ecosystem that is in peril.     

Some findings of FADD knockdown in inhibition of HIV-1 replication in Jurkat cells and PBMCs

Fas-associated protein with death domain (FADD) is a key adaptor molecule transmitting the death signal mediated by death receptors, and it is also required for T cell proliferation. A recent study indicated that FADD is able to affect HIV-1 production, but the mechanism is not known. Using the susceptible Jurkat cell line and peripheral blood mononuclear cells, we studied the effects of FADD on HIV-1 production. TaqMan RT-PCR was used to quantify HIV-1 viral RNA copies, and Western blot analysis was used to detect protein expression. FADD knockdown decreased HIV-1 replication and inactivated caspase-3 activity in the cells and blocked CD4 translocation to the lipid rafts of the plasma membrane. Reduced expression of FADD suppressed TCR signaling through downregulation of TCR, CD3, and Zap-70 in response to HIV-1 infection and blocked the trafficking of TCR, CD3, CD28, and Zap-70 to lipid rafts, leading to reduced activation of NF-κB and NFAT, which are required for HIV-1 replication. FADD knockdown diminished caspase-8 migration to lipid rafts and its expression in response to HIV-1 infection. These results indicate that FADD, as a host pro-apoptotic protein, plays important roles in regulating HIV-1 replication and production in several ways, and apoptotic pathway inhibition is able to decrease HIV-1 replication and production.     

Genotypic Prediction of Tropism of Highly Diverse HIV-1 Strains from Cameroon

Background

The use of CCR5 antagonists involves determination of HIV-1 tropism prior to initiation of treatment. HIV-1 tropism can be assessed either by phenotypic or genotypic methods. Genotypic methods are extensively used for tropism prediction. However, their validation in predicting tropism of viral isolates belonging to group M non-B subtypes remains challenging. In Cameroon, the genetic diversity of HIV-1 strains is the broadest reported worldwide. To facilitate the integration of CCR5 antagonists into clinical practice in this region, there is a need to evaluate the performance of genotypic methods for predicting tropism of highly diverse group M HIV-1 strains.

Methods

Tropism of diverse HIV-1 strains isolated from PBMCs from Cameroon was determined using the GHOST cell assay. Prediction, based on V3 sequences from matched plasma samples, was determined using bioinformatics algorithms and rules based on position 11/25 and net charge applied independently or combined according to Delobel''s and Garrido''s rules. Performance of genotypic methods was evaluated by comparing prediction generated with tropism assigned by the phenotypic assay.

Results

Specificity for predicting R5-tropic virus was high, ranging from 83.7% to 97.7% depending on the genotypic methods used. Sensitivity for X4-tropic viruses was fairly low, ranging from 33.3% to 50%. In our study, overall, genotypic methods were less able to accurately predict X4-tropic virus belonging to subtype CRF02_AG. In addition, it was found that of the methods we used the Garrido rule has the highest sensitivity rate of over 50% with a specificity of 93%.

Conclusion

Our study demonstrated that overall, genotypic methods were less sensitive for accurate prediction of HIV-1 tropism in settings where diverse HIV-1 strains co-circulate. Our data suggest that further optimization of genotypic methods is needed and that larger studies to determine their utility for tropism prediction of diverse HIV-1 strains may be warranted.     

DNA Barcode Authentication of Wood Samples of Threatened and Commercial Timber Trees within the Tropical Dry Evergreen Forest of India

Background

India is rich with biodiversity, which includes a large number of endemic, rare and threatened plant species. Previous studies have used DNA barcoding to inventory species for applications in biodiversity monitoring, conservation impact assessment, monitoring of illegal trading, authentication of traded medicinal plants etc. This is the first tropical dry evergreen forest (TDEF) barcode study in the World and the first attempt to assemble a reference barcode library for the trees of India as part of a larger project initiated by this research group.

Methodology/Principal Findings

We sampled 429 trees representing 143 tropical dry evergreen forest (TDEF) species, which included 16 threatened species. DNA barcoding was completed using rbcL and matK markers. The tiered approach (1^st tier rbcL; 2^nd tier matK) correctly identified 136 out of 143 species (95%). This high level of species resolution was largely due to the fact that the tree species were taxonomically diverse in the TDEF. Ability to resolve taxonomically diverse tree species of TDEF was comparable among the best match method, the phylogenetic method, and the characteristic attribute organization system method.

Conclusions

We demonstrated the utility of the TDEF reference barcode library to authenticate wood samples from timber operations in the TDEF. This pilot research study will enable more comprehensive surveys of the illegal timber trade of threatened species in the TDEF. This TDEF reference barcode library also contains trees that have medicinal properties, which could be used to monitor unsustainable and indiscriminate collection of plants from the wild for their medicinal value.     

Simple sequence repeat marker development from bacterial artificial chromosome end sequences and expressed sequence tags of flax (Linum usitatissimum L.)

Flax is an important oilseed crop in North America and is mostly grown as a fibre crop in Europe. As a self-pollinated diploid with a small estimated genome size of ~370 Mb, flax is well suited for fast progress in genomics. In the last few years, important genetic resources have been developed for this crop. Here, we describe the assessment and comparative analyses of 1,506 putative simple sequence repeats (SSRs) of which, 1,164 were derived from BAC-end sequences (BESs) and 342 from expressed sequence tags (ESTs). The SSRs were assessed on a panel of 16 flax accessions with 673 (58 %) and 145 (42 %) primer pairs being polymorphic in the BESs and ESTs, respectively. With 818 novel polymorphic SSR primer pairs reported in this study, the repertoire of available SSRs in flax has more than doubled from the combined total of 508 of all previous reports. Among nucleotide motifs, trinucleotides were the most abundant irrespective of the class, but dinucleotides were the most polymorphic. SSR length was also positively correlated with polymorphism. Two dinucleotide (AT/TA and AG/GA) and two trinucleotide (AAT/ATA/TAA and GAA/AGA/AAG) motifs and their iterations, different from those reported in many other crops, accounted for more than half of all the SSRs and were also more polymorphic (63.4 %) than the rest of the markers (42.7 %). This improved resource promises to be useful in genetic, quantitative trait loci (QTL) and association mapping as well as for anchoring the physical/genetic map with the whole genome shotgun reference sequence of flax.     

Genome organisation and retrotransposon driven molecular evolution of the endosperm Hardness (Ha) locus in Triticum aestivum cv Glenlea

Wheat endosperm texture is controlled primarily by a locus (Ha), which comprises Gsp-1, Pina and Pinb genes encoding the so-called grain softness protein, puroindoline-a and puroindoline-b, respectively. Pina and Pinb were detected only on the D-genome of hexaploid wheat and its diploid progenitors while Gsp-1 was on all three homoeologous loci. Hexaploid cultivar Glenlea has a hard phenotype due to a null Pina genotype (D-genome) but the sequence organization is not reported. This study aimed at understanding the evolution of homoeologous Ha loci. Sequencing of three BAC clones from cv Glenlea was performed and sequence analyses delimited the Ha loci which spanned 3,925, 5,330 and 31,607 bp in the A-, B- and D-genomes, respectively. A solo LTR of Angela retroelement, downstream to Gsp-A1 and a fragment of Sabrina retroelement, downstream of Gsp-B1, were discovered. We propose that the insertion of these elements into the intergenic regions have driven the deletions of genomic segments harbouring Pina and Pinb genes in the A- and B-genomes of hexaploid wheat. Similarly, fragments of Romani and Vagabond retroelements were identified between truncated Pina and Pinb genes, indicating their role in the deletion of Pina in Glenlea, leading to its hard texture. Structural differences of the Ha locus region of the A-genome between two hexaploid wheat varieties namely Glenlea and Renan (CR626929), suggested the presence of more than one tetraploid ancestor in the origin of hexaploid wheat.