Conventional digital cameras as a tool for assessing leaf area index and biomass for cereal breeding

Affordable and easy-to-use methods for assessing biomass and leaf area index （LAI） would be of interest in most breeding programs. Here, we describe the evaluation of a protocol for photographic sampling and image analysis aimed at providing low-labor yet robust indicators of biomass and LAI. In this trial, two genotypes of triticale, two of bread wheat, and four of tritordeum were studied. At six dates during the growing cycle, biomass and LAI were measured destructively, and digital photography was taken on the same dates. Several vegetation indices were calculated from each image. The results showed that repeatable and consistent values of the indices were obtained in consecutive photographic samplings on the same plots. The photographic indices were highly correlated with the destructive measure-ments, though the magnitude of the correlation was lower after anthesis. This work shows that photographic assess-ment of biomass and LAI can be fast, affordable, have good repeatability, and can be used under bright and overcast skies. A practical vegetation index derived from pictures is the fraction of green pixels over the total pixels of the image, and as it shows good correlations with all biomass variables, is the most robust to lighting conditions and has easy interpretation.     

Flow cytometric detection and quantification of heterotrophic nanoflagellates in enriched seawater and cultures

A flow cytometric protocol to detect and enumerate heterotrophic nanoflagellates (HNF) in enriched waters is reported. At present, the cytometric protocols that allow accurate quantification of bacterioplankton cannot be used to quantify protozoa for the following reasons: i) the background produced by the bacterial acquisitions does not allow the discrimination of protozoa at low abundance, ii) since the final protozoan fluorescence is much higher than the bacterioplankton fluorescence (more than 35 fold) the protozoa acquisitions lie outside the range. With an increase in the fluorescence threshold and a reduction of the fluorescence detector voltage, low fluorescence particles (bacteria) are beneath the detection limits and only higher fluorescence particles (most of them heterotrophic nanoflagellates) are detected. The main limitation for the application of the cytometric protocol developed is that a ratio of bacteria/HNF below 1000 is needed. At higher ratios, the background of larger cells of bacterioplankton makes it difficult to discriminate protozoa. The proposed protocol has been validated by epifluorescence microscopy analyzing both a mixed community and two single species of HFN: Rhynchomonas nasuta and Jakoba libera. Taking into account the required bacteria/HNF ratio cited above, the results provide evidence that the flow cytometric protocol reported here is valid for counting mixed communities of HNF in enriched seawater and in experimental micro or mesocosms. In the case of single species of HNF previous knowledge of the biological characteristics of the protist and how they can affect the effectiveness of the flow cytometric count is necessary.     

Applying Microsatellite Multiplex PCR Analysis (MMPA) for Determining Allele Copy-Number Status and Percentage of Normal Cells within Tumors

The study of somatic genetic alterations in tumors contributes to the understanding and management of cancer. Genetic alterations, such us copy number or copy neutral changes, generate allelic imbalances (AIs) that can be determined using polymorphic markers. Here we report the development of a simple set of calculations for analyzing microsatellite multiplex PCR data from control-tumor pairs that allows us to obtain accurate information not only regarding the AI status of tumors, but also the percentage of tumor-infiltrating normal cells, the locus copy-number status and the mechanism involved in AI. We validated this new approach by re-analyzing a set of Neurofibromatosis type 1-associated dermal neurofibromas and comparing newly generated data with results obtained for the same tumors in a previous study using MLPA, Paralog Ratio Analysis and SNP-array techniques.Microsatellite multiplex PCR analysis (MMPA) should be particularly useful for analyzing specific regions of the genome containing tumor suppressor genes and also for determining the percentage of infiltrating normal cells within tumors allowing them to be sorted before they are analyzed by more expensive techniques.     

The Péclet effect on leaf water enrichment correlates with leaf hydraulic conductance and mesophyll conductance for CO(2)

Leaf water gets isotopically enriched through transpiration, and diffusion of enriched water through the leaf depends on transpiration flow and the effective path length (L). The aim of this work was to relate L with physiological variables likely to respond to similar processes. We studied the response to drought and vein severing of leaf lamina hydraulic conductance (K_lamina), mesophyll conductance for CO₂ (g_m) and leaf water isotope enrichment in Vitis vinifera L cv. Grenache. We hypothesized that restrictions in water pathways would reduce K_lamina and increase L. As a secondary hypothesis, we proposed that, given the common pathways for water and CO₂ involved, a similar response should be found in g_m. Our results showed that L was strongly related to mesophyll variables, such as K_lamina or g_m across experimental drought and vein‐cutting treatments, showing stronger relationships than with variables included as input parameters for the models, such as transpiration. Our findings were further supported by a literature survey showing a close link between L and leaf hydraulic conductance (K_leaf = 31.5 × L^?0.43, r² = 0.60, n = 24). The strong correlation found between L, K_lamina and g_m supports the idea that water and CO₂ share an important part of their diffusion pathways through the mesophyll.     

Prognostic value of X-chromosome inactivation in symptomatic female carriers of dystrophinopathy

Background

Neonatal screening for Pompe disease has been introduced in Taiwan and a few U.S. states, while other jurisdictions including some European countries are piloting or considering this screening. First-tier screening flags both classic infantile and late-onset Pompe disease, which challenges current screening criteria. Previously, advocacy groups have sometimes supported expanded neonatal screening more than professional experts, while neutral citizens' views were unknown. This study aimed to measure support for neonatal screening for Pompe disease in the general public and to compare it to support among (parents of) patients with this condition. The study was done in the Netherlands, where newborns are not currently screened for Pompe disease. Newborn screening is not mandatory in the Netherlands but current uptake is almost universal.

Methods

A consumer panel (neutral group) and (parents of) patients with Pompe disease (Pompe group) were sent information and a questionnaire. Responses were analyzed of 555 neutral and 58 Pompe-experienced informants who had demonstrated sufficient understanding.

Results

87% of the neutral group and 88% of the Pompe group supported the introduction of screening (95% CI of difference -10 to 7%). The groups were similar in their moral reasoning about screening and acceptance of false positives, but the Pompe-experienced group expected greater benefit from neonatal detection of late-onset disease. Multivariate regression analysis controlling for demographics confirmed that approval of the introduction of screening was independent of having (a child with) Pompe disease. Furthermore, respondents with university education, regardless of whether they have (a child with) Pompe disease, were more likely to be reluctant about the introduction of screening than those with less education, OR for approval 0.29 (95% CI 0.18 to 0.49, p < 0.001).

Conclusions

This survey suggests a rather high level of support for newborn screening for Pompe disease, not only among those who have personal experience of the disease but also among the general public in the Netherlands. Optional screening on the basis of informed parental consent is probably unrealistic, underlining the need for new guidelines to help policymakers in their consideration of newborn screening for broad phenotype conditions.     

The dawn of human matrilineal diversity

The quest to explain demographic history during the early part of human evolution has been limited because of the scarce paleoanthropological record from the Middle Stone Age. To shed light on the structure of the mitochondrial DNA (mtDNA) phylogeny at the dawn of Homo sapiens, we constructed a matrilineal tree composed of 624 complete mtDNA genomes from sub-Saharan Hg L lineages. We paid particular attention to the Khoi and San (Khoisan) people of South Africa because they are considered to be a unique relic of hunter-gatherer lifestyle and to carry paternal and maternal lineages belonging to the deepest clades known among modern humans. Both the tree phylogeny and coalescence calculations suggest that Khoisan matrilineal ancestry diverged from the rest of the human mtDNA pool 90,000-150,000 years before present (ybp) and that at least five additional, currently extant maternal lineages existed during this period in parallel. Furthermore, we estimate that a minimum of 40 other evolutionarily successful lineages flourished in sub-Saharan Africa during the period of modern human dispersal out of Africa approximately 60,000-70,000 ybp. Only much later, at the beginning of the Late Stone Age, about 40,000 ybp, did introgression of additional lineages occur into the Khoisan mtDNA pool. This process was further accelerated during the recent Bantu expansions. Our results suggest that the early settlement of humans in Africa was already matrilineally structured and involved small, separately evolving isolated populations.     

Orymold: ontology based gene expression data integration and analysis tool applied to rice

Background  

Integration and exploration of data obtained from genome wide monitoring technologies has become a major challenge for many bioinformaticists and biologists due to its heterogeneity and high dimensionality. A widely accepted approach to solve these issues has been the creation and use of controlled vocabularies (ontologies). Ontologies allow for the formalization of domain knowledge, which in turn enables generalization in the creation of querying interfaces as well as in the integration of heterogeneous data, providing both human and machine readable interfaces.     

Probing the binding mechanism and affinity of tanezumab, a recombinant humanized anti-NGF monoclonal antibody, using a repertoire of biosensors

We describe the use of four complementary biosensors (Biacore 3000, Octet QK, ProteOn XPR36, and KinExA 3000) in characterizing the kinetics of human nerve growth factor (NGF) binding to a humanized NGF-neutralizing monoclonal antibody (tanezumab, formerly known as RN624). Tanezumab is a clinical candidate as a therapy for chronic pain. Our measurements were consistent with the NGF/tanezumab binding affinity being tighter than 10 pM due to the formation of an extremely stable complex that had an estimated half-life exceeding 100 h, which was beyond the resolution of any of our methods. The system was particularly challenging to study because NGF is an obligate homodimer, and we describe various assay orientations and immobilization methods that were used to minimize avidity in our experiments while keeping NGF in as native a state as possible. We also explored the interactions of NGF with its natural receptors, TrkA and P75, and how tanezumab blocks them. The Biacore blocking assay that we designed was used to quantify the potency of tanezumab and is more precise and reproducible than the currently available cell-based functional assays.