User-Friendly Multifunctional Red-Emissive Carbon Dots for Rapid Cell Nucleus Staining via Targeting Nuclear Proteins.

Cytoarchitectural staining is of great importance in disease diagnosis and cell biology research. This study developed user-friendly multifunctional red-emissive carbon dots (R-CDs) for rapid cell nucleus staining via targeting nuclear proteins. R-CDs, simply prepared by electrochemical treatment of 1,2,4-benzenetriamine, exhibit strong emission at 635 nm when excited at 507 nm. The R-CDs can rapidly stain the nucleus of human SH-SY5Y, HepG2, and HUH-7 cells with a high signal-to-noise ratio owing to fluorescence enhancement after entering the nucleus. Compared to conventional cytosolic dyes such as Hoechst and DAPI, R-CDs are cheaper, more highly dispersed in water, and more stable (requiring no stringent storage conditions). The R-CDs show stable optical properties with insignificant photobleaching over 7 days and salt resistance up to 2 M of NaCl. More importantly, R-CDs, possessing a positive charge, allow rapid staining of live cells (3 min) and dead cells (10 s) in saline. According to kinetic variation, R-CDs can distinguish live cells from dead cells. Staining exhibits high efficiency in onion epidermal cells, Aspergillus niger, Caenorhabditis elegans, and human spermatozoa. The mechanism for efficient staining is based on their fast accumulation in the nucleus due to their small size and positive charge and strong interaction with nuclear proteins at amino acid residues of histidine and arginine, resulting in fluorescence enhancement by dozens of times. The developed R-CDs do not bind to DNA and would not cause genetic damage and will find various safe applications in biological and medical fields.

Dynamic Structural Change of Plant Epidermal Cell Walls under Strain.

The molecular foundations of epidermal cell wall mechanics are critical for understanding structure-function relationships of primary cell walls in plants and facilitating the design of bioinspired materials. To uncover the molecular mechanisms regulating the high extensibility and strength of the cell wall, the onion epidermal wall is stretched uniaxially to various strains and cell wall structures from mesoscale to atomic scale are characterized. Upon longitudinal stretching to high strain, epidermal walls contract in the transverse direction, resulting in a reduced area. Atomic force microscopy shows that cellulose microfibrils exhibit orientation-dependent rearrangements at high strains: longitudinal microfibrils are straightened out and become highly ordered, while transverse microfibrils curve and kink. Small-angle X-ray scattering detects a 7.4 nm spacing aligned along the stretch direction at high strain, which is attributed to distances between individual cellulose microfibrils. Furthermore, wide-angle X-ray scattering reveals a widening of (004) lattice spacing and contraction of (200) lattice spacing in longitudinally aligned cellulose microfibrils at high strain, which implies longitudinal stretching of the cellulose crystal. These findings provide molecular insights into the ability of the wall to bear additional load after yielding: the aggregation of longitudinal microfibrils impedes sliding and enables further stretching of the cellulose to bear increased loads.

Rice amino acid transporter-like 6 (OsATL6) is involved in amino acid homeostasis by modulating the vacuolar storage of glutamine in roots.

Glutamine is a product of ammonium (NH4+ ) assimilation catalyzed by glutamine synthetase (GS) and glutamate synthase (GOGAT). The growth of NH4+ -preferring paddy rice (Oryza sativa L.) depends on root NH4+ assimilation and the subsequent root-to-shoot allocation of glutamine; however, little is known about the mechanism of glutamine storage in roots. Here, using transcriptome and reverse genetics analyses, we show that the rice amino acid transporter-like 6 (OsATL6) protein exports glutamine to the root vacuoles under NH4+ -replete conditions. OsATL6 was expressed, along with OsGS1;2 and OsNADH-GOGAT1, in wild-type (WT) roots fed with sufficient NH4 Cl, and was induced by glutamine treatment. We generated two independent Tos17 retrotransposon insertion mutants showing reduced OsATL6 expression to determine the function of OsATL6. Compared with segregants lacking the Tos17 insertion, the OsATL6 knock-down mutant seedlings exhibited lower root glutamine content but higher glutamine concentration in the xylem sap and greater shoot growth under NH4+ -replete conditions. The transient expression of monomeric red fluorescent protein-fused OsATL6 in onion epidermal cells confirmed the tonoplast localization of OsATL6. When OsATL6 was expressed in Xenopus laevis oocytes, glutamine efflux from the cell into the acidic bath solution increased. Under sufficient NH4+ supply, OsATL6 transiently accumulated in sclerenchyma and pericycle cells, which are located adjacent to the Casparian strip, thus obstructing the apoplastic solute path, and in vascular parenchyma cells of WT roots before the peak accumulation of GS1;2 and NADH-GOGAT1 occurred. These findings suggest that OsATL6 temporarily stores excess glutamine, produced by NH4+ assimilation, in root vacuoles before it can be translocated to the shoot.

Single-shot multi-depth full-field optical coherence tomography using spatial frequency division multiplexing.

Fast 3D volumetric imaging has been essential for biology, medicine and industrial inspections, and various optical coherence tomography (OCT) methods have been developed to meet such needs. Point-scanning based approaches, such as swept-source OCT and spectral domain OCT, can obtain a depth information at once, but they require lateral scan for full 3D imaging. On the contrary, full-field OCT needs the scanning of imaging depth while it records a full lateral information at once. Here, we present a full-field OCT system that can obtain multi-depth information at once by a single-shot recording. We combine a 2D diffraction grating and a custom-made echelon to prepare multiple reference beams having different pathlengths and propagating angles. By recording a single interference image between the reflected wave from a sample and these multiple reference beams, we reconstruct full-field images at multiple depths associated with the pathlengths of the individual reference beams. We demonstrated the single-shot recording of 7 different depth images at 10 µm for biological tissues. Our method can potentially be useful for applications where high-speed recording of multiple en-face images is crucial.

A highly effective "turn-on" camphor-based fluorescent probe for rapid and sensitive detection and its biological imaging of Fe2.

In this work, a novel fluorescent probe CBO was synthesized for detecting Fe2+ using the natural monoterpenketone camphor as the starting material. The probe CBO displayed turn-on fluorescence to Fe2+ accompanied by the solution change from colorless to green. As expected, there was an excellent linear relationship between the fluorescence intensity of probe CBO and the concentration of Fe2+ (0-20 µM), and the detection limit was as low as 1.56×10-8 M. In particular, CBO could selectively sense Fe2+ more than other analytes (Fe3+ included) through the N-oxide strategy, and quickly responded to Fe2+ (60 s) over a wide pH (4-14) range. Additionally, based on the rapid fluorescence response of CBO to Fe2+, a simple test strip-based detector was designed for boosting practical applicability. The probe CBO had been successfully applied to the fluorescence imaging of Fe2+ in onion cells and living zebrafish. The probe CBO was a powerful tool of detecting Fe2+ level in organisms, which was of significance to understand the role of Fe2+ in Fe2+-related physical processes and diseases.

Cytomembrane visualization using Stokes parameter confocal microscopy.

A new, to the best of our knowledge, method for Stokes vector imaging is proposed to achieve imaging and dynamic monitoring of a non-labeled cytomembrane. In this work, a polarization state vector is described by a Stokes vector and expressed in chrominance space. A physical quantity called polarization chromaticity value (PCV) corresponding to a Stokes vector is used as the imaging parameter to perform Stokes vector imaging. By using the PCV imaging technique, the Stokes vector can be expressed in three-dimensional real space rather than in a Poincare sphere. Furthermore, a four-way Stokes parameter confocal microscopy system is designed to measure four Stokes parameters simultaneously and obtain micro-imaging. Label-free living onion cell membranes and their plasmolysis process are selected as the representative micro-anisotropy experimental analysis. It is proved that PCV imaging can perform visualization of cytomembranes, and further, microscopic orientation is demonstrated. The prospect of universal measurement of anisotropy details for analysis and diagnosis is provided.

Real-time quantitative phase imaging by single-shot dual-wavelength off-axis digital holographic microscopy.

A single-shot dual-wavelength digital holographic microscopy with an adjustable off-axis configuration is presented, which helps realize real-time quantitative phase imaging for living cells. With this configuration, two sets of interference fringes corresponding to their wavelengths can be flexibly recorded onto one hologram in one shot. The universal expression on the dual-wavelength hologram recorded under any wave vector orientation angles of reference beams is given. To avoid as much as possible the effect of zero-order spectrum, we can flexibly select their carry frequencies for the two wavelengths using this adjustable off-axis configuration, according to the distribution feature of object's spatial-frequency spectrum. This merit is verified by a quantitative phase imaging experiment for the microchannel of a microfluidic chip. The reconstructed phase maps of living onion epidermal cells exhibit cellular internal life activities, for the first time to the best of our knowledge, vividly displaying the progress of the nucleus, cell wall, cytoskeleton, and the substance transport in microtubules inside living cells. These imaging results demonstrate the availability and reliability of the presented method for real-time quantitative phase imaging.

Evaluation of toxicological and antimicrobial activity of lavender and immortelle essential oils.

Lavender and immortelle essential oils (EOs) are widely used to treat a spectrum of human conditions. The aim of this study was to investigate cyto/genotoxic effects of lavender and immortelle EOs using plant cells (Allium cepa) and human lymphocytes, as well as their antimicrobial potential using nine strains of bacteria and fungi. Our results for lavender and immortelle EOs showed that the frequency of chromosome aberrations (CAs) was increased in comparison with controls. For both oils, increased frequency of apoptosis for all concentrations, as well as the frequency of necrosis (0.10/0.30 µl/ml for lavender/immortelle, respectively) was demonstrated. In human lymphocytes, differences for minute fragments between immortelle oil (0.10 µl/ml) and controls were observed. Increased frequency of apoptosis was detected for immortelle oil (0.20 µl/ml), while both oils (0.20; 0.30 µl/ml lavender, and immortelle at all concentrations) induced higher frequency of necrosis in comparison with controls. Lavender EO was effective against all tested Gram-positive and Gram-negative bacteria, while immortelle EO inhibited only Gram-positive bacteria. Both oils exhibited antifungal effect. Our results demonstrated that lavender and immortelle EOs showed cyto/genotoxic effects in both, plant and human cells, as well as antimicrobial properties. Further studies are needed to strengthen these findings.

The MIR-Domain of PbbHLH2 Is Involved in Regulation of the Anthocyanin Biosynthetic Pathway in "Red Zaosu" (PyrusBretschneideri Rehd.) Pear Fruit.

The N-terminal of Myc-like basic helix-loop-helix transcription factors (bHLH TFs) contains an interaction domain, namely the MYB-interacting region (MIR), which interacts with the R2R3-MYB proteins to regulate genes involved in the anthocyanin biosynthetic pathway. However, the functions of MIR-domain bHLHs in this pathway are not fully understood. In this study, PbbHLH2 containing the MIR-domain was identified and its function investigated. The overexpression of PbbHLH2 in "Zaosu" pear peel increased the anthocyanin content and the expression levels of late biosynthetic genes. Bimolecular fluorescence complementation showed that PbbHLH2 interacted with R2R3-MYB TFs PbMYB9, 10, and 10b in onion epidermal cells and confirmed that MIR-domain plays important roles in the interaction between the MIR-domain bHLH and R2R3-MYB TFs. Moreover, PbbHLH2 bound and activated the dihydroflavonol reductase promoter in yeast one-hybrid (Y1H) and dual-luciferase assays. Taken together these results suggested that the MIR domain of PbbHLH2 regulated anthocyanin biosynthesis in pear fruit peel.

Quantum-inspired detection for spectral domain optical coherence tomography.

Intensity levels allowed by safety standards (ICNIRP or ANSI) limit the amount of light that can be used in a clinical setting to image highly scattering or absorptive tissues with optical coherence tomography (OCT). To achieve high-sensitivity imaging at low intensity levels, we adapt a detection scheme-which is used in quantum optics for providing information about spectral correlations of photons-into a standard spectral domain OCT system. This detection scheme is based on the concept of dispersive Fourier transformation, where a fiber introduces a wavelength-dependent time delay measured by a single-pixel detector, usually a high-speed photoreceiver. Here, we use a fast superconducting single-photon detector SSPD as a single-pixel detector and obtain images of a glass stack and a slice of onion at the intensity levels of the order of 10 pW. We also provide a formula for a depth-dependent sensitivity falloff in such a detection scheme, which can be treated as a temporal equivalent of diffraction-grating-based spectrometers.

5-Aminouracil and other inhibitors of DNA replication induce biphasic interphase-mitotic cells in apical root meristems of Allium cepa.

KEY MESSAGE: Induction of biphasic interphase-mitotic cells and PCC is connected with an increased level of metabolism in root meristem cells of Allium cepa. Previous experiments using primary roots of Allium cepa exposed to low concentrations of hydroxyurea have shown that long-term DNA replication stress (DRS) disrupts essential links of the S-M checkpoint mechanism, leading meristem cells either to premature chromosome condensation (PCC) or to a specific form of chromatin condensation, establishing biphasic organization of cell nuclei with both interphase and mitotic domains (IM cells). The present study supplements and extends these observations by describing general conditions under which both abnormal types of M-phase cells may occur. The analysis of root apical meristem (RAM) cell proliferation after prolonged mild DRS indicates that a broad spectrum of inhibitors is capable of generating PCC and IM organization of cell nuclei. These included: 5-aminouracil (5-AU, a thymine antagonist), characterized by the highest efficiency in creating cells with the IM phenotype, aphidicolin (APH), an inhibitor of DNA polymerase α, 5-fluorodeoxyuridine (FUdR), an inhibitor of thymidylate synthetase, methotrexate (MTX), a folic acid analog that inhibits purine and pyrimidine synthesis, and cytosine arabinoside (Ara-C), which inhibits DNA replication by forming cleavage complexes with topoisomerase I. As evidenced using fluorescence-based click chemistry assays, continuous treatment of onion RAM cells with 5-AU is associated with an accelerated dynamics of the DNA replication machinery and significantly enhanced levels of transcription and translation. Furthermore, DRS conditions bring about an intensified production of hydrogen peroxide (H2O2), depletion of reduced glutathione (GSH), and some increase in DNA fragmentation, associated with only a slight increase in apoptosis-like programmed cell death events.

Comparative cyto- and genotoxicity of 900 MHz and 1800 MHz electromagnetic field radiations in root meristems of Allium cepa.

In the last few decades, tremendous increase in the use of wireless electronic gadgets, particularly the cell phones, has significantly enhanced the levels of electromagnetic field radiations (EMF-r) in the environment. Therefore, it is pertinent to study the effect of these radiations on biological systems including plants. We investigated comparative cytotoxic and DNA damaging effects of 900 and 1800 MHz EMF-r in Allium cepa (onion) root meristematic cells in terms of mitotic index (MI), chromosomal aberrations (CAs) and single cell gel electrophoresis (comet assay). Onion bulbs were subjected to 900 and 1800 MHz (at power densities 261 ±â€¯8.50 mW m-2 and 332 ±â€¯10.36 mW m-2, respectively) of EMF-r for 0.5 h, 1 h, 2 h, and 4 h. Root length declined by 13.2% and 12.3%, whereas root thickness was increased by 46.7% and 48.3% after 4 h exposure to 900 MHz and 1800 MHz, respectively. Cytogenetic studies exhibited clastogenic effect of EMF-r as depicted by increased CAs and MI. MI increased by 36% and 53% after 2 and 4 h exposure to 900 MHz EMF-r, whereas it increased by 41% and 67% in response to 1800 MHz EMF-r. Aberration index was increased by 41%-266% and 14%-257% during 0.5-4 h of exposure to 900 MHz and 1800 MHz, respectively, over the control. EMF-r exposure decreased % head DNA (DNAH) and increased % tail DNA (DNAT) and olive tail moment (OTM) at both 900 and 1800 EMF-r. In 4 h exposure treatments, head DNA (%) declined by 19% and 23% at 900 MHz and 1800 MHz, respectively. DNAT and OTM were increased by 2.3 and 3.7 fold upon exposure to 900 MHz EMF-r over that in the control, whereas 2.8 and 5.8 fold increase was observed in response to 1800 MHz EMF-r exposure for 4 h and the difference was statistically significant. The study concludes that EMF-r in the communication range (900 and 1800 MHz) adversely affect root meristems in plants and induce cytotoxic and DNA damage. EMF-r induced DNA damage was more pronounced at 1800 MHz than that at 900 MHz.

Application of cytogenetic model Allium cepa for screening potential cytogenotoxicity of herbal-based hair dyes.

Plant models may be useful as test organisms for initial screening of potential toxicity of personal care products. The objective of the present study was to assess the efficacy of the Allium cepa (common onion) test system as a bioanalytical tool for screening potential cytotoxicity and genotoxicity of herbal-based hair dye formulations. Exposure of black hair dye formulations for 48 hours resulted in root growth retardation and mitosis suppression in the root meristems of A. cepa bulbs indicating concentration dependent cytotoxicity. At the 72 hour post exposure, cytotoxic effects on the roots were reduced but not recovered completely signifying prolong toxic action of the hair dyes. The condensed nuclei was the most frequent nuclear abnormality found in the dye exposed root meristematic cells indicating the cell death process. Induction of micronuclei and chromosomal aberrations in the root meristematic cells even at the post exposure stage indicates persistent genotoxicity of the hair dyes which may be attributed to the interactive effects of chemical mixtures present in the commercial hair dye formulations. The results revealed that A. cepa test system is an effective bioanalytical tool for screening cytogenotoxicity of commercial hair dye formulations.

Treated Textile Effluents: Cytotoxic and Genotoxic Effects in the Natural Aquatic Environment.

Textile effluent treatment methods use biological and chemical treatments to reduce the toxicity and to comply with standard effluent discharge limits. However, trace amounts of pollutants can affect the biological organisms in the receiving environment. The present study used Allium cepa bio assay to assess the cytotoxic and genotoxic effects of treated textile effluents discharged to the natural environment. The results of the bioassay indicated that treated textile effluents can induce alterations in the mitotic index. Also nuclear buds, bi nuclei, condensed nuclei, were recorded in the bioassay and the severity of them decreased towards downstream of the effluent discharge point. Therefore, it can be concluded that even the discharged effluents comply with the standard limits, there is a possibility of causing cytotoxic and genotoxic effects in the organisms living in the natural environment.

Cytotoxic and Genotoxic Assessment of Silicon Dioxide Nanoparticles by Allium and Comet Tests.

Silicon nanoparticles gained a great interest due to its use in biomedical research. It is considered as safe and has been used in nanomedicine. But literature still states its toxicity depending upon the size and dose of silicon nanoparticles. So, current study was aimed to evaluate the cytotoxicity and genotoxicity of silicon dioxide nanoparticles (SiO2NPs) by Allium anaphase-telophase and Comet tests. Characterization of SiO2NPs showed the particle size as 16.12 ± 3.07 nm. The mean diameter of SiO2NPs was having range of 404.66 ± 93.39 nm in solution. Highest total anomalies (18.80 ± 0.45) were observed at 100 µg/mL, whereas least (11.2 ± 0.84) were observed by the 12.5 µg/mL concentration. There was concentration-response association in increased CAs and DNA damage. The highest concentration (100 µg/mL) of SiO2NPs induced the significant DNA damage (149.67 ± 1.15), whereas the least was observed by the negative control (2.67 ± 0.58). The current study revealed the cytotoxic and genotoxic effects of SiO2NPs on the root meristem cells of A. cepa.

Quantitative Analysis of Metabolites at the Single-Cell Level by Hydrogen Flame Desorption Ionization Mass Spectrometry.

To date, direct quantitation of cellular metabolites at the picoliter level or in a single cell is still a challenge due to tiny sampling materials, the accuracy of the sampling volume, and the ubiquitous matrix effect. Herein, picoliter magnitude quantitative analysis was performed using a pressure-assisted microsampling probe coupled to the hydrogen flame desorption ionization mass spectrometer (HFDI-MS). The sampling was accurately controlled with a picoliter pump, and the analytes were rapidly vaporized and quantitatively transferred to the gas phase by adequate heat. The vapor-phase analytes reacted with protonated water cluster ions by the proton-transfer reaction (PTR). The accurate sampling, flash thermal desorption, and proton-transfer ionization processes were conducted spatiotemporally, which could greatly reduce matrix effects to facilitate the quantitation of analytes without the internal standard. Furthermore, this workflow enabled the quantitation of cellular metabolites at the picoliter/single-cell level.

Long-term holographic phase-contrast time lapse reveals cytoplasmic circulation in dehydrating plant cells.

The intracellular dynamics of onion epidermal cells during the dehydration process is observed by holographic microscopy. Both the nucleus and cytoplasm are accurately revealed by quantitative phase imaging while dehydration takes place. Indeed, we notice that the contrast of phase images increases with the decrease in cellular water content. We foresee that such a dehydrating process can be effective for improving phase contrast, thus permitting better imaging of plant cells with the scope of learning more about cellular dynamics and related phenomena. Exploiting this concept, we observe intracellular cytoplasmic circulation and transport of biological material.

Nucleolar alterations are reliable parameters to determine the cytogenotoxicity of environmental pollutants.

Pollution generated by deposition of industrial activity waste in the environment without due care can lead to serious environmental consequences. Bioassays in higher plants are means of understanding the cytogenotoxic effects of these substances. In the present work, Allium cepa L. was used as a model species to assess nucleolar changes induced by environmental pollutants. The substances used were Methyl Methane Sulfonate (MMS), cadmium (Cd), Spent Potliner (SPL) and the herbicide Atrazine. Water was used as a negative control. The silver-stained nucleolar organizer region (AgNOR) assay was used making it possible to evaluate how nucleolar parameters (number of nucleoli per nucleus and nucleoli area) behave when facing stress caused by such pollutants. The results obtained showed a variation in the observed parameters: an increase in the number of nucleoli in the treated cells and tendency to a reduction in nucleolar area, indicating that the tested pollutants may have impaired nucleolar activity. In addition, it was possible to establish a relationship between the behavior of the nucleolus with other changes as plantlet growth, cell proliferation, and DNA damage.

Molecular Cloning and Functional Identification of a Squalene Synthase Encoding Gene from Alfalfa (Medicago sativa L.).

The quality of alfalfa, a main forage legume worldwide, is of great importance for the dairy industry and is affected by the content of triterpene saponins. These natural terpenoid products of triterpene aglycones are catalyzed by squalene synthase (SQS), a highly conserved enzyme present in eukaryotes. However, there is scare information on alfalfa SQS. Here, an open reading frame (ORF) of SQS was cloned from alfalfa. Sequence analysis showed MsSQS had the same exon/intron composition and shared high homology with its orthologs. Bioinformatic analysis revealed the deduced MsSQS had two transmembrane domains. When transiently expressed, GFP-MsSQS fusion protein was localized on the plasma membrane of onion epidermal cells. Removal of the C-terminal transmembrane domain of MsSQS improved solubility in Escherichia coli. MsSQS was preferably expressed in roots, followed by leaves and stems. MeJA treatment induced MsSQS expression and increased the content of total saponins. Overexpression of MsSQS in alfalfa led to the accumulation of total saponins, suggesting a correlation between MsSQS expression level with saponins content. Therefore, MsSQS is a canonical squalene synthase and contributes to saponin synthesis in alfalfa. This study provides a key candidate gene for genetic manipulation of the synthesis of triterpene saponins, which impact both plant and animal health.

Quantitative Extraction and Mass Spectrometry Analysis at a Single-Cell Level.

Quantitative live cell mass spectrometry analysis at a subcellular level requires the precisely controlled extraction of subpicoliter volumes of material from the cell, sensitive analysis of the extracted analytes, and their accurate quantification without prior separation. In this study, we demonstrate that localized electroosmotic extraction provides a direct path to addressing this challenge. Specifically, we demonstrate quantitative mass spectrometry analysis of biomolecules in picoliter volumes extracted from live cells. Electroosmotic extraction was performed using two electrodes and a finely pulled nanopipette with tip diameter of <1 µm containing a hydrophobic electrolyte compatible with mass spectrometry analysis. The electroosmotic drag was used to drive analytes out of the cell into the nanopipette. Analyte molecules extracted both from solutions and cell samples were analyzed using nanoelectrospray ionization (nanoESI) directly from the nanopipette into a mass spectrometer. More than 50 metabolites including sugars and flavonoids were detected in positive mode in 2-5 pL volumes of the cytoplasmic material extracted from Allium cepa. Quantification of the extracted glucose was performed using sequential extraction of a known volume of the aqueous solution containing glucose- d2 standard of known concentration. We found that the ratio of the signal of glucose to glucose- d2 increased linearly with glucose concentration. This observation indicates that the approach developed in this study enables quantitative analysis of small volumes of metabolites extracted from cells. Furthermore, we observed efficient separation of hydrophilic and hydrophobic analytes through partitioning into the aqueous and hydrophobic electrolyte phase, respectively, which provides additional important information on the molecular properties of extracted metabolites.