Morphology and chemical composition of Taiwan oil millet (Eccoilopus formosanus) epicuticular wax.

MAIN CONCLUSION: Taiwan oil millet has two types of epicuticular wax: platelet wax composed primarily of octacosanol and filament wax constituted essentially by the singular compound of octacosanoic acid. Taiwan oil millet (TOM-Eccoilopus formosanus) is an orphan crop cultivated by the Taiwan indigenous people. It has conspicuous white powder covering its leaf sheath indicating abundant epicuticular waxes, that may contribute to its resilience. Here, we characterized the epicuticular wax secretion in TOM leaf blade and leaf sheath using various microscopy techniques, as well as gas chromatography to determine its composition. Two kinds of waxes, platelet and filaments, were secreted in both the leaf blades and sheaths. The platelet wax is secreted ubiquitously by epidermal cells, whereas the filament wax is secreted by a specific cell called epidermal cork cells. The newly developed filament waxes were markedly re-synthesized by the epidermal cork cells through papillae protrusions on the external periclinal cell wall. Ultrastructural images of cork cell revealed the presence of cortical endoplasmic reticulum (ER) tubules along the periphery of plasma membrane (PM) and ER-PM contact sites (EPCS). The predominant wax component was a C28 primary alcohol in leaf blade, and a C28 free fatty acid in the leaf sheath, pseudopetiole and midrib. The wax morphology present in distinct plant organs corresponds to the specific chemical composition: platelet wax composed of alcohols exists mainly in the leaf blade, whereas filament wax constituted mainly by the singular compound C28 free fatty acids is present abundantly in leaf sheath. Our study clarifies the filament wax composition in relation to a previous study in sorghum. Both platelet and filament waxes comprise a protection barrier for TOM.

Rice GA3ox1 modulates pollen starch granule accumulation and pollen wall development.

The rice GA biosynthetic gene OsGA3ox1 has been proposed to regulate pollen development through the gametophytic manner, but cellular characterization of its mutant pollen is lacking. In this study, three heterozygotic biallelic variants, "-3/-19", "-3/-2" and "-3/-10", each containing one null and one 3bp-deletion allele, were obtained by the CRISPR/Cas9 technique for the functional study of OsGA3ox1. The three homozygotes, "-19/-19", "-2/-2" and "-10/-10", derived from heterozygotic variants, did not affect the development of most vegetative and floral organs but showed a significant reduction in seed-setting rate and in pollen viability. Anatomic characterizations of these mutated osga3ox1 pollens revealed defects in starch granule accumulation and pollen wall development. Additional molecular characterization suggests that abnormal pollen development in the osga3ox1 mutants might be linked to the regulation of transcription factors OsGAMYB, OsTDR and OsbHLH142 during late pollen development. In brief, the rice GA3ox1 is a crucial gene that modulates pollen starch granule accumulation and pollen wall development at the gametophytic phase.

Simultaneous detection of miRNA and mRNA at the single-cell level in plant tissues.

Detecting the simultaneous presence of a microRNA (miRNA) and a mRNA in a specific tissue can provide support for the prediction that the miRNA regulates the mRNA. Although two such methods have been developed for mammalian tissues, they have a low signal-noise ratio and/or poor resolution at the single-cell level. To overcome these drawbacks, we develop a method that uses sequence-specific miRNA-locked nucleic acid (LNA) and mRNA-LNA probes. Moreover, it augments the detection signal by rolling circle amplification, achieving a high signal-noise ratio at the single-cell level. Dot signals are counted for determining the expression levels of mRNA and miRNA molecules in specific cells. We show a high sequence specificity of our miRNA-LNA probe, revealing that it can discriminate single-base mismatches. Numerical quantification by our method is tested in transgenic rice lines with different gene expression levels. We conduct several applications. First, the spatial expression profiling of osa-miR156 and OsSPL12 in rice leaves reveals their specific expression in mesophyll cells. Second, studying rice and its mutant lines with our method reveals opposite expression patterns of miRNA and its target mRNA in tissues. Third, the dynamic expression profiles of ZmGRF8 and zma-miR396 during maize leaf development provide evidence that zma-miR396 regulates the preferential spatial expression of ZmGRF8 in bundle sheath cells. Finally, our method can be scaled up to simultaneously detect multiple miRNAs and mRNAs in a tissue. Thus, it is a sensitive and versatile technique for studying miRNA regulation of plant tissue development.

Determinants of genetic variation across eco-evolutionary scales in pinnipeds.

The effective size of a population (Ne), which determines its level of neutral variability, is a key evolutionary parameter. Ne can substantially depart from census sizes of present-day breeding populations (NC) as a result of past demographic changes, variation in life-history traits and selection at linked sites. Using genome-wide data we estimated the long-term coalescent Ne for 17 pinniped species represented by 36 population samples (total n = 458 individuals). Ne estimates ranged from 8,936 to 91,178, were highly consistent within (sub)species and showed a strong positive correlation with NC ([Formula: see text] = 0.59; P = 0.0002). Ne/NC ratios were low (mean, 0.31; median, 0.13) and co-varied strongly with demographic history and, to a lesser degree, with species' ecological and life-history variables such as breeding habitat. Residual variation in Ne/NC, after controlling for past demographic fluctuations, contained information about recent population size changes during the Anthropocene. Specifically, species of conservation concern typically had positive residuals indicative of a smaller contemporary NC than would be expected from their long-term Ne. This study highlights the value of comparative population genomic analyses for gauging the evolutionary processes governing genetic variation in natural populations, and provides a framework for identifying populations deserving closer conservation attention.

Development of wearable posture monitoring system for dynamic assessment of sitting posture.

There have been increasing cases of people seeking treatment for neck and back pain. The most common cause of neck and back pain is due to long-term poor sitting posture. The most common poor sitting posture cases are humpback, and head and neck being too far forward. It is easy to cause neck and back pain and other symptoms. Therefore, the development of wearable posture monitoring system for dynamic assessment of sitting posture becomes both helpful and necessary. In addition to recording the wearer's posture when sitting with quantitative assessment, it is needed to execute real-time action feedback for correctness of posture, in order to reduce neck and back pain due to long-term poor sitting posture. This study completed an instant recording and dynamic assessment of position measurement and feedback system. The system consists of a number of dynamic measurement units that can describe the posture trajectory, which integrates three-axis gyro meter, three-axis accelerometer, and magnetometer in order to measure the dynamic tracking. In the reliability analysis experiment, angle measurement error is less than 2%. The correlation coefficient between correlation analysis and Motion Analysis (MA) is 0.97. It is shown that the motion trajectory of this system is highly correlated with MA. In the feasibility test of sitting position detection, it is possible to detect the sitting position from the basic action of the walking, standing, sitting and lying down, and the sensitivity reaches 95.84%. In the assessment of the sitting position, the information published by the Canadian Centre for Occupational Health and Safety was used, as well as the recommendations of professional physicians as a basis for evaluating the threshold of the sitting measurement parameters and immediately feedback to the subjects. The system developed in this study can be helpful to reduce neck and back pain due to long-term poor sitting posture.

In situ quantification of individual mRNA transcripts in melanocytes discloses gene regulation of relevance to speciation.

Functional validation of candidate genes involved in adaptation and speciation remains challenging. Here, we exemplify the utility of a method quantifying individual mRNA transcripts in revealing the molecular basis of divergence in feather pigment synthesis during early-stage speciation in crows. Using a padlock probe assay combined with rolling circle amplification, we quantified cell-type-specific gene expression in the histological context of growing feather follicles. Expression of Tyrosinase Related Protein 1 (TYRP1), Solute Carrier Family 45 member 2 (SLC45A2) and Hematopoietic Prostaglandin D Synthase (HPGDS) was melanocyte-limited and significantly reduced in follicles from hooded crow, explaining the substantially lower eumelanin content in grey versus black feathers. The central upstream Melanocyte Inducing Transcription Factor (MITF) only showed differential expression specific to melanocytes - a feature not captured by bulk RNA-seq. Overall, this study provides insight into the molecular basis of an evolutionary young transition in pigment synthesis, and demonstrates the power of histologically explicit, statistically substantiated single-cell gene expression quantification for functional genetic inference in natural populations.

Chiral liquid chromatography-tandem mass spectrometry assay to determine that dexpramipexole is not converted to pramipexole in vivo after administered in humans.

Dexpramipexole (DEX) was being investigated in clinical studies for the treatment of amyotrophic lateral sclerosis (ALS). To monitor the potential chiral interconversion of dexpramipexole to pramipexole (PPX) in vivo, a highly sensitive and selective chiral LC-MS/MS assay was developed and qualified for the detection of pramipexole in the presence of dexpramipexole in human plasma. In this assay, plasma samples were extracted by protein precipitation coupled with solid phase extraction (SPE). The analyte PPX was separated from its enantiomer DEX using a chiral HPLC method. The assay was qualified with a dynamic range of 0.150-1.00ng/mL. The lower limit of quantitation (LLOQ) for PPX was 0.150ng/mL in the presence of up to 1000ng/mL of DEX. The qualified method was used to analyze plasma samples from a DEX clinical study. No PPX was detected in humans at pharmacologically significant levels after administration of dexpramipexole at single doses up to 600mg per day.

Kin recognition within a seed and the effect of genetic relatedness of an endosperm to its compatriot embryo on maize seed development.

As one of two sexual products resulting from double fertilization in angiosperms, the endosperm nourishes its compatriot embryo during seed development and/or germination and ultimately dies. Theoretical studies suggest that the genetic relatedness of an endosperm to its embryo in the same seed might determine the amount of resources ultimately available for the embryo during seed development. We took advantage of the phenomenon of heterofertilization in cultivated maize to empirically test whether genetic relatedness between a triploid embryo-nourishing endosperm and its compatriot diploid embryo impacts the process of resource allocation between these two sexually produced entities. We used genetically distinct maize inbred lines to perform two crossing experiments. Dry weights of dissected embryos and endosperms of mature heterofertilized and adjacent homofertilized kernels were compared. Embryo weight of heterofertilized kernels was significantly less than that of embryos of homofertilized kernels, whereas there was no significant difference in endosperm weight between the two types of kernels. Our results suggest that the degree of genetic relatedness of an endosperm to its compatriot embryo affects seed development and specifically the amount of maternal resources allocated to an endosperm that are eventually turned over to an embryo. The lower the coefficient of relatedness of an endosperm to its compatriot embryo, the smaller the embryo. Thus, the endosperm of a heterofertilized seed appears to behave less cooperatively with respect to resource transfer toward its less closely related embryo compared with the endosperm of a homofertilized seed.

Female gametophyte development and double fertilization in Balsas teosinte, Zea mays subsp. parviglumis (Poaceae).

Over the course of maize evolution, domestication played a major role in the structural transition of the vegetative and reproductive characteristics that distinguish it from its closest wild relative, Zea mays subsp. parviglumis (Balsas teosinte). Little is known, however, about impacts of the domestication process on the cellular features of the female gametophyte and the subsequent reproductive events after fertilization, even though they are essential components of plant sexual reproduction. In this study, we investigated the developmental and cellular features of the Balsas teosinte female gametophyte and early developing seed in order to unravel the key structural and evolutionary transitions of the reproductive process associated with the domestication of the ancestor of maize. Our results show that the female gametophyte of Balsas teosinte is a variation of the Polygonum type with proliferative antipodal cells and is similar to that of maize. The fertilization process of Balsas teosinte also is basically similar to domesticated maize. In contrast to maize, many events associated with the development of the embryo and endosperm appear to be initiated earlier in Balsas teosinte. Our study suggests that the pattern of female gametophyte development with antipodal proliferation is common among species and subspecies of Zea and evolved before maize domestication. In addition, we propose that the relatively longer duration of the free nuclear endosperm phase in maize is correlated with the development of a larger fruit (kernel or caryopsis) and with a bigger endosperm compared with Balsas teosinte.

Evolution of Lecythidaceae with an emphasis on the circumscription of neotropical genera: information from combined ndhF and trnL-F sequence data.

The Lecythidaceae comprise a pantropical family best known for the edible seeds of the Brazil nut (Bertholletia excelsa) and the cannon-ball tree (Couroupita guianensis), which is planted as a botanical curiosity in subtropical and tropical gardens. In addition, species of the family are often among the most common in neotropical forests, especially in the Amazon Basin. The Brazil nut family is diverse and abundant in the Amazon and is considered to be an indicator of undisturbed or scarcely disturbed lowland forests; thus, what is learned about its evolution, ecology, and biogeography may suggest similar patterns for other Amazonian tree families. We used combined data sets derived from the ndhF and trnL-F genes to elucidate relationships of genera in both the Old and New Worlds that have been associated with Lecythidaceae. Our molecular tree agrees with the recognition of Napoleonaeaceae and Scytopetalaceae. Within the Lecythidaceae, there is molecular support for recognizing three subfamilies: Foetidioideae, Planchonioideae, and Lecythidoideae. We then focused on genera of the Lecythidoideae and found support for recognizing Allantoma (when the actinomorphic-flowered species of Cariniana are included in it), Grias, Gustavia, Corythophora, Couratari, and Couroupita, but conclude that Cariniana, Lecythis, and Eschweilera are not monoyphyletic. Because the position of the monotypic Bertholletia excelsa in relation to the other zygomorphic-flowered genera is not resolved, we are not able to comment on its generic relationships.

Protein spray freeze drying. 2. Effect of formulation variables on particle size and stability.

Spray freeze drying produces protein particles suitable for microencapsulation into polymeric microspheres intended for sustained release. Accessibility of encapsulated protein particles to the microsphere surface increases as the protein particle size is increased. Thus, it is desirable that the encapsulated protein particle size be minimized to limit initial release. We have investigated the effect of formulation on spray freeze-dried bovine serum albumin (BSA) as a model protein. Atomization conditions were fixed such that in the absence of excipient, the particle size of the sonicated powder was submicron, and there was substantial protein degradation (loss of monomer). Addition of low concentrations of surfactants (up to the CMC) or mannitol (up to the point where it tended to crystallize upon dehydration) resulted in partial stabilization without impacting particle size. Trehalose was successful in stabilizing the protein; however, there was a marked increase in particle size at the highest levels tested. Ammonium sulfate provided partial stabilization, but also tended to form crystals and increase particle size. FTIR measurements showed a loss of native secondary structure upon spray freeze drying that was ameliorated by addition of trehalose. Other excipients did not prevent structural perturbations. In general, stabilization of spray freeze-dried BSA was related to lowering of the specific surface area in the powder. A balance must be achieved when spray freeze drying proteins intended for encapsulation in sustained-release systems.