Deep imaging reveals dynamics and signaling in one-to-one pollen tube guidance.

In the pistil of flowering plants, each ovule usually associates with a single pollen tube for fertilization. This one-to-one pollen tube guidance, which contributes to polyspermy blocking and efficient seed production, is largely different from animal chemotaxis of many sperms to one egg. However, the functional mechanisms underlying the directional cues and polytubey blocks in the depths of the pistil remain unknown.

The Chaperone NASP Contributes to de Novo Deposition of the Centromeric Histone Variant CENH3 in Arabidopsis Early Embryogenesis.

The centromere is an essential chromosome region where the kinetochore is formed to control equal chromosome distribution during cell division. The centromere-specific histone H3 variant CENH3 (also called CENP-A) is a prerequisite for the kinetochore formation. Since CENH3 evolves rapidly, associated factors, including histone chaperones mediating the deposition of CENH3 on the centromere, are thought to act through species-specific amino acid sequences.

Optical Clearing of Plant Tissues for Fluorescence Imaging.

It is challenging to directly observe the internal structure of multi-layered and opaque plant specimens, without dissection, under a microscope. In addition, autofluorescence attributed to chlorophyll hampers the observation of fluorescent proteins in plants. For a long time, various clearing reagents have been used to make plants transparent.

Biphasic electrochemical peptide synthesis.

The large amount of waste derived from coupling reagents is a serious drawback of peptide synthesis from a green chemistry viewpoint. To overcome this issue, we report an electrochemical peptide synthesis in a biphasic system. Anodic oxidation of triphenylphosphine (PhP) generates a phosphine radical cation, which serves as the coupling reagent to activate carboxylic acids, and produces triphenylphosphine oxide (PhP[double bond, length as m-dash]O) as a stoichiometric byproduct.

Hydrosilane-Mediated Electrochemical Reduction of Amides.

Electrochemical reduction of amides was achieved by using a hydrosilane without any toxic or expensive metals. The key reactive ketyl radical intermediate was generated by cathodic reduction. Continuous reaction with anodically generated silyl radicals or zinc bromide resulted in chemoselective deoxygenation to give the corresponding amines.

Detection of a biolistic delivery of fluorescent markers and CRISPR/Cas9 to the pollen tube.

Biolistic delivery into pollen. In recent years, genome editing techniques, such as the CRISPR/Cas9 system, have been highlighted as a new approach to plant breeding. Agrobacterium-mediated transformation has been widely utilized to generate transgenic plants by introducing plasmid DNA containing CRISPR/Cas9 into plant cells.

ClearSeeAlpha: Advanced Optical Clearing for Whole-Plant Imaging.

To understand how the body of plants is made, it is essential to observe the morphology, structure and arrangement of constituent cells. However, the opaque nature of the plant body makes it difficult to observe the internal structures directly under a microscope. To overcome this problem, we developed a reagent, ClearSee, that makes plants transparent, allowing direct observation of the inside of a plant body without inflicting damage on it, e.

Polyspermy Block in the Central Cell During Double Fertilization of .

During double fertilization in angiosperms, two male gametes (sperm cells), are released from a pollen tube into the receptive region between two female gametes; the egg cell and the central cell of the ovule. The sperm cells fertilize the egg cell and the central cell in a one-to-one manner to yield a zygote and an endosperm, respectively. The one-to-one distribution of the sperm cells to the two female gametes is strictly regulated, possibly via communication among the four gametes.

The male gamete membrane protein DMP9/DAU2 is required for double fertilization in flowering plants.

All flowering plants exhibit a unique type of sexual reproduction called 'double fertilization' in which each pollen tube-delivered sperm cell fuses with an egg and a central cell. Proteins that localize to the plasma membrane of gametes regulate one-to-one gamete pairing and fusion between male and female gametes for successful double fertilization. Here, we have identified a membrane protein from generative cells using proteomic analysis and have found that the protein is an ortholog of DUF679 DOMAIN MEMBRANE PROTEIN 9 (DMP9)/DUO1-ACTIVATED UNKNOWN 2 (DAU2).

Pollen differentiation as well as pollen tube guidance and discharge are independent of the presence of gametes.

After meiosis, an unequal cell division generates the male gamete lineage in flowering plants. The generative cell will undergo a second division, giving rise to the two gametes, i.e.

Pollen tube contents initiate ovule enlargement and enhance seed coat development without fertilization.

In angiosperms, pollen tubes carry two sperm cells toward the egg and central cells to complete double fertilization. In animals, not only sperm but also seminal plasma is required for proper fertilization. However, little is known regarding the function of pollen tube content (PTC), which is analogous to seminal plasma.

Generation of a homozygous fertilization-defective gcs1 mutant by heat-inducible removal of a rescue gene.

Key message: New gametic homozygous mutants. In angiosperms, a haploid male gamete (sperm cell) fuses with a haploid female gamete (egg cell) during fertilization to form a zygote carrying paternally and maternally derived chromosomes. Several fertilization-defective mutants in Arabidopsis thaliana, including a generative cell-specific 1 (gcs1)/hapless 2 mutant, the sperm cells of which are unable to fuse with female gametes, can only be maintained as heterozygous lines due to the infertile male or female gametes.

Bloch simulations towards direct detection of oscillating magnetic fields using MRI with spin-lock sequence.

A new MRI method using the spin-lock sequence has attracted wide attention because of its potential for detecting small oscillating magnetic fields. However, as the mechanism involved is complicated, we visualized the magnetization performance during the spin-lock sequence in order to better understand interaction of the spin-lock pulse and the externally applied oscillating magnetic fields by means of a fast-and-simple method using matrix operations to solve a time-dependent Bloch equation. To improve spin-lock imaging in the detection of small magnetic fields (in an fMRI experiment that modeled neural magnetic fields), we observed that the phenomenon decreases MR signals, which led us to investigate how spin-lock parameters cause the MR signal to decrease; based on this, we determined that MR signals decrease in oscillating magnetic fields that are resonant with the spin-lock pulse.

Double fertilization on the move.

Double fertilization is a flowering plant mechanism whereby two immotile sperm cells fertilize two different female gametes. One of the two sperm cells fertilizes the egg cell to produce the embryo and the other fertilizes the central cell to produce the endosperm. Despite the biological and agricultural significance of double fertilization, the mechanism remains largely unknown owing to difficulties associated with the embedded structure of female gametes in the maternal tissue.

Atelocollagen-mediated systemic delivery prevents immunostimulatory adverse effects of siRNA in mammals.

Short interfering RNA (siRNA) is a potent activator of the mammalian innate immune system. When considering possible clinical applications of siRNA for humans, the adverse immunostimulatory effects must also be taken into account. Here, we show that atelocollagen-mediated systemic delivery of siRNA without chemical modifications did not cause any immunostimulation in both animals and human peripheral blood mononuclear cells (PBMCs), even if the siRNA harbored an interferon (IFN)-inducible sequence.

Acquisition parameters for diffusion tensor imaging to emphasize fractional anisotropy: phantom study.

Diffusion tensor imaging (DTI) is a magnetic resonance (MR) imaging technique that has attracted attention in recent years for applications such as nerve fiber tracking, neurography, and tumor detection. In DTI measurements, 2 motion-probing gradient (MPG) pulses are applied to evaluate water diffusion. In DTI for nerve fiber tracking, acquisition parameters, such as strength, duration, and separation of MPGs, influence the MR signal.

Systemic delivery of siRNA specific to tumor mediated by atelocollagen: combined therapy using siRNA targeting Bcl-xL and cisplatin against prostate cancer.

The largest obstacle to the effective use of short interfering RNA (siRNA) in an animal body is the ability to deliver it to the target tissue. Here we showed a systemic delivery method of siRNA specific to pregrown solid tumors via atelocollagen. Atelocollagen facilitated the selective uptake of siRNA into the tumors when an siRNA/atelocollagen complex was administered intravenously to mice.

RPN2 gene confers docetaxel resistance in breast cancer.

Drug resistance acquired by cancer cells has led to treatment failure. To understand the regulatory network underlying docetaxel resistance in breast cancer cells and to identify molecular targets for therapy, we tested small interfering RNAs (siRNAs) against 36 genes whose expression was elevated in human nonresponders to docetaxel for the ability to promote apoptosis of docetaxel-resistant human breast cancer cells (MCF7-ADR cells). The results indicate that the downregulation of the gene encoding ribophorin [corrected] II (RPN2), which is part of an N-oligosaccharyl transferase complex, most efficiently induces apoptosis of MCF7-ADR cells in the presence of docetaxel.

Downregulation of monocyte chemoattractant protein-1 involving short interfering RNA attenuates hapten-induced contact hypersensitivity.

Contact hypersensitivity (CHS) is a common skin disease, presenting clinically as allergic contact dermatitis. At inflammatory sites in a typical CHS model in the mouse ear, elevated expression of monocyte chemoattractant protein-1 (MCP-1) has been reported. MCP-1 is a potent chemotactic factor for many types of leukocytes including monocytes/macrophages and T cells.

Single-injection ornithine decarboxylase-directed antisense therapy using atelocollagen to suppress human cancer growth.

Background: Substantial evidence supports a direct role of ornithine decarboxylase (ODC) in the development and maintenance of human tumors. Although antisense oligonucleotide therapy targeting various genes are useful for cancer treatment, 1 of the major limitations is the problem of delivery. A novel antisense oligonucleotide delivery method is described that allows prolonged sustainment and release of ODC antisense oligonucleotides in vivo using atelocollagen.

Atelocollagen-mediated drug discovery technology.

RNA interference (RNAi) was first reported in nematodes in 1998. Since that time, RNAi has been discovered in fish, insects and mammals. Novel treatments and drug discovery in preclinical studies based on RNAi are targeting a wide range of diseases at present, including viral infections and cancers.

Atelocollagen-mediated systemic DDS for nucleic acid medicines.

The goal of our research is to provide a practical platform for drug delivery in oligonucleotide therapy. We report here the efficacy of an atelocollagen-mediated oligonucleotide delivery system applied to systemic siRNA and antisense oligonucleotide treatments in animal disease models. Atelocollagen and oligonucleotides formed a complex of nanosized particles, which was highly stable against nucleases.

Efficient delivery of small interfering RNA to bone-metastatic tumors by using atelocollagen in vivo.

Silencing of gene expression by small interfering RNAs (siRNAs) is rapidly becoming a powerful tool for genetic analysis and represents a potential strategy for therapeutic product development. However, there are no reports of systemic delivery for siRNAs toward treatment of bone-metastatic cancer. Accordingly, we report here that i.