Development of a seed DNA-based genotyping system for marker-assisted selection in maize

Leaf collection from the field, labeling and tracking back to the source plants after genotyping are rate limiting steps in leaf DNA-based genotyping. In this study, an optimized genotyping method using endosperm DNA sampled from single maize seeds was developed, which can be used to replace leaf DNA-based genotyping for both genetic studies and breeding applications. A similar approach is likely to be suitable for all plants with relatively large seeds. Part of the endosperm was excised from imbibed maize seeds and DNA extracted in 96-tube plates using individuals from eight F₂ populations and seven inbreds. The quality of the resultant DNA was functionally comparable to DNA extracted from leaf tissue. Extraction from 30 mg of endosperm yields 3–10 μg DNA, which is sufficient for analysis of 200–400 agarose-gel PCR-based markers, with the potential for several million chip-based SNP marker analyses. By comparing endosperm DNA and leaf DNA for individuals from an F₂ population, genotyping errors caused by pericarp contamination and hetero-fertilization were found to average 3.8 and 0.6%, respectively. Endosperm sampling did not affect germination rates under controlled conditions, although under normal field conditions the germination rate, seedling establishment, and growth vigor were significantly lower than that of non-sampled controls for some genotypes. However, careful field management can compensate for these effects. Seed DNA-based genotyping lowered costs by 24.6% compared to leaf DNA-based genotyping due to reduced field plantings and labor costs. A substantial advantage of this approach is that it can be used to select desirable genotypes before planting. As such it provides an opportunity for dramatic improvements in the efficiency and selective gain of breeding systems based on optimum combinations of marker-assisted selection and phenotypic selection within and between generations.     

Improving the accuracy of a solid spherical source radius and depth estimation using the diffusion equation in fluorescence reflectance mode

Background  

Non-invasive planar fluorescence reflectance imaging (FRI) is used for accessing physiological and molecular processes in biological tissue. This method is efficiently used to detect superficial fluorescent inclusions. FRI is based on recording the spatial radiance distribution (SRD) at the surface of a sample. SRD provides information for measuring structural parameters of a fluorescent source (such as radius and depth). The aim of this article is to estimate the depth and radius of the source distribution from SRD, measured at the sample surface. For this reason, a theoretical expression for the SRD at the surface of a turbid sample arising from a spherical light source embedded in the sample, was derived using a steady-state solution of the diffusion equation with an appropriate boundary condition.     

Selenite and ebselen supplementation attenuates <Emphasis Type="SmallCaps">d</Emphasis>-galactose-induced oxidative stress and increases expression of SELR and SEP15 in rat lens

Selenite and ebselen supplementation has been shown to possess anti-cataract potential in some experimental animal models of cataract, however, the underlying mechanisms remain unclear. The present study was designed to evaluate the anti-cataract effects and the underlying mechanisms of selenite and ebselen supplementation on galactose induced cataract in rats, a common animal model of sugar cataract. Transmission electron microscopy images of lens fiber cells (LFC) and lens epithelial cells (LEC) were observed in d-galactose-induced experimental cataractous rats treated with or without selenite and ebselen, also redox homeostasis and expression of proteins such as selenoprotein R (SELR), 15kD selenoprotein (SEP15), superoxide dismutase 1 (SOD1), catalase (CAT), β-crystallin protein, aldose reductase (AR) and glucose-regulated protein 78 (GRP78) were estimated in the lenses. The results showed that d-galactose injection injured rat lens and resulted in cataract formation; however, selenite and ebselen supplementation markedly alleviated ultrastructural injury of LFC and LEC. Moreover, selenite and ebselen supplementation could mitigate the oxidative damage in rat lens and increase the protein expressions of SELR, SEP15, SOD1, CAT and β-crystallin, as well as decrease the protein expressions of AR and GRP78. Taken together, these findings for the first time reveal the anti-cataract potential of selenite and ebselen in galactosemic cataract, and provide important new insights into the anti-cataract mechanisms of selenite and ebselen in sugar cataract.     

Landscape genetics of a tropical rescue pollinator

Pollination services are increasingly threatened by the loss and modification of natural habitats, posing a risk to the maintenance of both native plant biodiversity and agricultural production. In order to safeguard pollination services, it is essential to examine the impacts of habitat degradation on the population dynamics of key pollinators and identify potential “rescue pollinators” capable of persisting in these human-altered landscapes. Using a landscape genetic approach, we assessed the impact of landscape structure on genetic differentiation in the widely-distributed tropical stingless bee Trigona spinipes (Apidae: Meliponini) across agricultural landscape mosaics composed of coffee plantations and Atlantic forest fragments in southeastern Brazil. We genotyped 115 bees at 16 specific and highly polymorphic microsatellite loci, developed using next-generation sequencing. Our results reveal that T. spinipes is capable of dispersing across remarkably long distances, as we did not find genetic differentiation across a 200 km range, nor fine-scale spatial genetic structure. Furthermore, gene flow was not affected by forest cover, land cover, or elevation, indicating that reproductive individuals are able to disperse well through agricultural landscapes and across altitudinal gradients. We also found evidence of a recent population expansion, suggesting that this opportunistic stingless bee is capable of colonizing degraded habitats. Our results thus suggest that T. spinipes can persist in heavily-altered landscapes and can be regarded as a rescue pollinator, potentially compensating for the decline of other native pollinators in degraded tropical landscapes.     

Engineering bacteria toward tumor targeting for cancer treatment: current state and perspectives

One of the primary limitations of cancer therapy is lack of selectivity of therapeutic agents to tumor cells. Current efforts are focused on discovering and developing anticancer agents that selectively target only tumor cells and spare normal cells to improve the therapeutic index. The use of preferentially replicating bacteria as an oncolytic agent is one of the innovative approaches for the treatment of cancer. This is based on the observation that some obligate or facultative anaerobic bacteria are capable of multiplying selectively in tumors and inhibiting their growth. Meanwhile, bacteria have been demonstrated to colonize and destroy tumor, and have emerged as biological gene vectors to tumor microenvironment. To improve the efficacy and safety of the bacterial therapy, a further understanding of bacteria between with immune system is required. Furthermore, we want to evaluate how bacterial infection facilitates the “bystander effect” of chemotherapeutic agent and assess if it can be used for additional antitumor effect when combined with chemotherapy. This study may not only evaluate therapeutic efficacy of bacteria for the treatment of cancer but also elucidate the mechanisms underlying antitumor activities mediated by bacteria, which involve host immune responses and the cellular molecular responses.     

Immune suppression in the tumor microenvironment: a role for dendritic cell-mediated tolerization of T cells

Immune suppression remains a consistent obstacle to successful anti-tumor immune responses. As tumors develop, they create a microenvironment that not only supports tumor growth and metastasis but also reduces potential adaptive immunity to tumor antigens. Among the many components of this tumor microenvironment is a population of dendritic cells which exert profound immune suppressive effects on T cells. In this review, we discuss our recent findings related to these tumor-associated dendritic cells and how targeting them may serve to generate more durable anti-tumor immune responses.     

Synthesis of β-Peptide Standards for Use in Model Prebiotic Reactions

A one-pot method was developed for the preparation of a series of β-alanine standards of moderate size (2 to ≥12 residues) for studies concerning the prebiotic origins of peptides. The one-pot synthesis involved two sequential reactions: (1) dry-down self-condensation of β-alanine methyl ester, yielding β-alanine peptide methyl ester oligomers, and (2) subsequent hydrolysis of β-alanine peptide methyl ester oligomers, producing a series of β-alanine peptide standards. These standards were then spiked into a model prebiotic product mixture to confirm by HPLC the formation of β-alanine peptides under plausible reaction conditions. The simplicity of this approach suggests it can be used to prepare a variety of β-peptide standards for investigating differences between α- and β-peptides in the context of prebiotic chemistry.     

Differential expression analysis for sequence count data

High-throughput sequencing assays such as RNA-Seq, ChIP-Seq or barcode counting provide quantitative readouts in the form of count data. To infer differential signal in such data correctly and with good statistical power, estimation of data variability throughout the dynamic range and a suitable error model are required. We propose a method based on the negative binomial distribution, with variance and mean linked by local regression and present an implementation, DESeq, as an R/Bioconductor package.     

Chromogranin A and the Tumor Microenvironment

Chromogranin A (CgA) is an acidic glycoprotein belonging to a family of regulated secretory proteins stored in the dense core granules of the adrenal medulla and of many other neuroendocrine cells and neurons. This protein is frequently used as a diagnostic and prognostic serum marker for a range of neuroendocrine tumors. Circulating CgA is also increased in patients with other diseases, including subpopulations of patients with non-neuroendocrine tumors, with important prognostic implications. A growing body of evidence suggests that CgA is more than a diagnostic/prognostic marker for cancer patients. Indeed, results of in vitro experiments and in vivo studies in animal models suggest that this protein and its fragments can affect several elements of the tumor microenvironment, including fibroblasts and endothelial cells. In this article, recent findings implicating CgA as a modulator of the tumor microenvironment and suggesting that abnormal secretion of CgA could play important roles in tumor progression and response to therapy in cancer patients are reviewed and discussed.     

Analysis of rhamnolipid biosurfactants by methylene blue complexation

Rhamnolipids, produced by Pseudomonas aeruginosa, represent an important group of biosurfactants having various industrial, environmental, and medical applications. Current methods for rhamnolipid quantification involve the use of strong hazardous acids/chemicals, indirect measurement of the concentration of sugar moiety, or require the availability of expensive equipment (HPLC-MS). A safer, easier method that measures the whole rhamnolipid molecules would significantly enhance strain selection, metabolic engineering, and process development for economical rhamnolipid production. A semi-quantitative method was reported earlier to differentiate between the rhamnolipid-producing and non-producing strains using agar plates containing methylene blue and cetyl trimethylammonium bromide (CTAB). In this study, a rapid and simple method for rhamnolipid analysis was developed by systematically investigating the complexation of rhamnolipids and methylene blue, with and without the presence of CTAB. The method relies on measuring the absorbance (at 638 nm) of the rhamnolipid−methylene blue complex that partitions into the chloroform phase. With P. aeruginosa fermentation samples, the applicability of this method was verified by comparison of the analysis results with those obtained from the commonly used anthrone reaction technique.