The male germ unit of Rhododendron : quantitative cytology, three-dimensional reconstruction, isolation and detection using fluorescent probes

The sperm cells of Rhododendron laetum and R. macgregoriae differentiate within the pollen tube about 24 h after germination in vitro. Threedimensional reconstruction shows that the sperm cells are paired together, and both have extensions that link with the tube nucleus, forming a male germ unit. Quantitative analysis shows that the sperm cells in each pair differ significantly in surface area, but not in cell volume nor in numbers of mitochondria or plastids. When isolated from pollen tubes by osmotic shock, the sperm cells became ellipsoidal and surrounded by their own plasma membrane, while a proportion remained in pairs linked by the inner tube plasma membrane. Both generative and sperm cells are visualized in pollen tube preparations by immunofluorescence with anti-tubulin and anti-actin monoclonal antibodies (MAbs) combined with H33258 fluorescence of the nuclei. Video-image processing shows the presence of an axial microtubule cage in the generative cells, and some microtubules are present in the cytoplasmic extensions that clasp the tube nucleus. Following sperm cell division, the extensive phragmoplast between the sperm nuclei is partitioned by the plasma membranes.

A fresh look at the validity of the diffusion approximation for modeling fluorescence spectroscopy in biological tissue

Fluorescence has become a widely used technique for applications in noninvasive diagnostic tissue spectroscopy. The standard model used for characterizing fluorescence photon transport in biological tissue is based on the diffusion approximation. On the premise that the total energy of excitation and fluorescent photon flows must be conserved, we derive the widely used diffusion equations in fluorescence spectroscopy and show that there must be an additional term to account for the transport of fluorescent photons. The significance of this additional term in modeling fluorescence spectroscopy in biological tissue is assessed.

Lawsone : a novel reagent for the detection of latent fingermarks on paper surfaces

Lawsone (2-hydroxy-1,4-naphthoquinone) reacts with latent fingermark deposits on paper surfaces to yield purple-brown impressions of ridge details which are also photoluminescent; this compound represents the first in a completely new class of fingermark detection reagents.

Surface-Enhanced Infrared Spectra of Manganese (III) Tetraphenylporphine Chloride Physisorbed on Gold Island Films

Surface-enhanced infrared absorption (SEIRA) spectra of manganese (III) tetraphenylporphine chloride (Mn(TPP)Cl) on metal island films were measured in transmission mode. Dependences of the enhancement factor of SEIRA on both the sample quantity and the type of evaporated metal were investigated by subsequently increasing the amount of Mn(TPP)Cl on gold and silver substrates. The enhancement increases nonlinearly with the amount of sample and varies slightly with the thickness of metal islands. In particular, the SEIRA transmission method presents an anomalous spectral enhancement by a factor of 579, with substantial spectral shifts, observed only for the physisorbed Mn(TPP)Cl that remained on a 3-nm-thick gold film after immersion of the substrates into acetone. A charge-transfer (CT) interaction between the porphyrinic Mn and gold islands is therefore proposed as an additional factor in the SEIRA mechanism of the porphyrin system. The number of remaining porphyrin molecules was estimated by calibration-based fluorescence spectroscopy to be 2.36×1013 molecules (i.e., ~2.910-11 mol/cm2) for a 3-nm-thick gold film, suggesting that the physisorbed molecules distributed very loosely on the metal island surface as a result of the weak van der Waals interactions. Fluorescence microscopy revealed the formation of microcrystalline porphyrin aggregates during the consecutive increase in sample solution. However, the immersion likely redistributed the porphyrin to be directly attached on the gold surface, as evidenced by an absence of porphyrinic microcrystals and the observed SEIRA enhancement. The distinctive red shift in the UV-visible spectra and the SEIRA-enhanced peaks indicate the presence of a preferred orientation in the form of the porphyrin ring inclined with respect to the gold surface.

Graphene/tri-block copolymer composites prepared via RAFT polymerizations for dual controlled drug delivery via pH stimulation and biodegradation

A novel tri-block copolymer poly(oxopentanoate ethyl methacrylate)-block-poly(pyridyl disulfide ethyl acrylate)-block-poly(ethylene glycol acrylate) [poly(OEMA-b-PDEA-b-PEGA)], retaining active keto groups and pyridyl disulfide (PDS) side functionalities, was synthesized as a drug delivery vehicle using reversible addition-fragmentation chain transfer (RAFT) polymerization method. One mimic drug pyridine-2-thione (PT) was introduced into the monomer, PDEA for copolymerization. The other mimic drug O-benzylhydroxylamine (BHA) was conjugated with tri-block copolymer via efficient oxime coupling chemistry, followed by the attachment onto graphene via π-π stacking interaction to obtain a graphene/tri-block copolymer composite. 1H NMR, UV-vis absorption spectroscopy, fluorescence spectroscopy, gel permeation chromatography (GPC), atomic force microscope (AFM) and transmission electron microscope (TEM) were used to verify the successful step-wise preparation of the tri-block copolymer and drug loaded composite. In vitro release behaviors of BHA and PT from graphene/tri-block copolymer composite via dual drug release mechanisms were investigated. BHA can be released under acid environment, while PT will be released in the presence of reducing agents, such as dithiothreitol (DTT) or glutathione (GSH). It can be envisioned that this novel composite could be exploited as a novel intracellular drug delivery system via dual release mechanisms.

Mechanical properties of graphene films enhanced by homo-telechelic functionalized polymer fillers via π-π Stacking interactions

© 2015 Elsevier Ltd. All rights reserved. Most researches on graphene/polymer composites are focusing on improving the mechanical and electrical properties of polymers at low graphene content instead of paying attention to constructing graphene's macroscopic structures. In current study the homo-telechelic functionalized polyethylene glycols (FPEGs) were tailored with π-orbital-rich groups (namely phenyl, pyrene and di-pyrene) via esterification reactions, which enhanced the interaction between polyethylene glycol (PEG) molecules and chemical reduced graphene oxide (RGO) sheets. The π-π stacking interactions between graphene sheets and π-orbital-rich groups endowed the composite films with enhanced tensile strength and tunable electrical conductivity. The formation of graphene network structure mediated by the FPEGs fillers via π-π stacking non-covalent interactions should account for the experimental results. The experimental investigations were also complemented with theoretical calculation using a density functional theory. Atomic force microscope (AFM), scanning electron microscope (SEM), X-ray diffraction (XRD), nuclear magnetic resonance (NMR), thermal gravimetric analysis (TGA), UV-vis and fluorescence spectroscopy were used to monitor the step-wise preparation of graphene composite films.

Synthesis of N-substituted 4-hydroxynaphthalimides using palladium-catalysed hydroxylation

Successful implementation of a palladium-mediated hydroxylation of N-substituted 4-chloro-1,8-naphthalimides has been achieved. The methodology detailed herein utilises 4-chloro-1,8-naphthalic anhydride as a starting point and implements the catalyst/ligand combination of Pd(OAc)2/t-BuXPhos; all of which are relatively inexpensive. A range of imide substituents tolerated the reaction conditions, including acid sensitive substrates which are not compatible with other existing methodology. As such this approach is not only complimentary to existing procedures, it presents a more direct alternative to accessing 4-hydroxy-1,8-naphthalimides.

Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm

Background
Rice is the primary source of food for billions of people in developing countries, yet the commonly consumed polished grain contains insufficient levels of the key micronutrients iron (Fe), zinc (Zn) and Vitamin A to meet daily dietary requirements. Experts estimate that a rice-based diet should contain 14.5 µg g−1 Fe in endosperm, the main constituent of polished grain, but breeding programs have failed to achieve even half of that value. Transgenic efforts to increase the Fe concentration of rice endosperm include expression of ferritin genes, nicotianamine synthase genes (NAS) or ferritin in conjunction with NAS genes, with results ranging from two-fold increases via single-gene approaches to six-fold increases via multi-gene approaches, yet no approach has reported 14.5 µg g−1 Fe in endosperm.

Methodology/Principal Findings
Three populations of rice were generated to constitutively overexpress OsNAS1, OsNAS2 or OsNAS3, respectively. Nicotianamine, Fe and Zn concentrations were significantly increased in unpolished grain of all three of the overexpression populations, relative to controls, with the highest concentrations in the OsNAS2 and OsNAS3 overexpression populations. Selected lines from each population had at least 10 µg g−1 Fe in polished grain and two OsNAS2 overexpression lines had 14 and 19 µg g−1 Fe in polished grain, representing up to four-fold increases in Fe concentration. Two-fold increases of Zn concentration were also observed in the OsNAS2 population. Synchrotron X-ray fluorescence spectroscopy demonstrated that OsNAS2 overexpression leads to significant enrichment of Fe and Zn in phosphorus-free regions of rice endosperm.

Conclusions
The OsNAS genes, particularly OsNAS2, show enormous potential for Fe and Zn biofortification of rice endosperm. The results demonstrate that rice cultivars overexpressing single rice OsNAS genes could provide a sustainable and genetically simple solution to Fe and Zn deficiency disorders affecting billions of people throughout the world.

Characterisation of a synthesised fluorescent ligand (4-acridinol-1-sulphonic acid) using nuclear magnetic resonance spectroscopy

The synthesis and complete characterisation of the fluorescent ligand, 4-acridinol-1-sulphonic acid (the acridine analogue of 8-quinolinol-5-sulfonic acid) is described. Using a judicious array of nuclear magnetic resonance spectroscopy experiments, the structural elucidation and full assignment of all proton and carbon chemical shifts were afforded. The 4-acridinol-1-sulphonic acid was found to behave in a similar manner to 8-quinolinol-5-sulphonic acid, forming fluorescent complexes with magnesium(II) and zinc(II). The uncorrected emission maxima for the metal–acridinol complexes were found to be at around 620 nm compared to 505 nm for the respective quinolinol complexes. Unfortunately, preliminary spectrofluorimetric analytical figures of merit revealed that the detection limits of the new acridinol metal complexes were one and a half orders of magnitude poorer than those attained with the corresponding quinolinol ligand. However, in contrast to 8-quinolinol-5-sulphonic acid, the 4-acridinol-1-sulphonic acid ligand showed considerable selectivity for magnesium(II) and zinc(II) over aluminium(III).

Fluorescent anion sensors based on 4-amino-1,8-naphthalimide that employ the 4-amino N-H

The new charge neutral 4-amino-1,8-naphthalimide based anion sensors 2 and 3 bind to both acetate and dihydrogenphosphate with 1:1 stoichiometry through hydrogen bonding to both thiourea N–H atoms and in the case of dihydrogenphosphate, the naphthalimide 4 amino N–H group as well. This was clearly established from ¹H NMR titration experiments with H₂PO₄^- in DMSO-d₆ where a substantial shift in the resonance for the naphthalimide N–H was observed concomitant with the expected migration of the thiourea N–H chemical shifts. The binding constants determined from the titration studies indicate that the new sensors bind H₂PO₄^- more strongly than AcO⁻. Fluorescence titrations with sensor 3 indicate quenching of 59% and 36% upon addition of acetate and dihydrogenphosphate, respectively.

Fluorescent photoinduced electron transfer (PET) sensors for anions; from design to potential application

This mini review highlights the synthesis and photophysical evaluation of anion sensors, for nonaqueous solutions, that have been developed in our laboratories over the last few years. We have focused our research mainly on developing fluorescent photoinduced electron transfer (PET) sensors based on the fluorophore-spacer-anion receptor principle using several anthracene (emitting in the blue) and 1,8-naphthalimide (emitting in the green) fluorophores, with the aim of targeting biologically and industrially relevant anions such as acetates, phosphate and amino acids, as well as halides such as fluoride. The receptors and the fluorophore are separated by a short methyl or ethyl spacer, where the charge neutral anion receptors are either aliphatic or aromatic urea (or thiourea) moieties. For these, the anion recognition is through hydrogen bonding, yielding anion:receptor complexes. Such bonding gives rise to enhanced reduction potential in the receptor moieties which causes enhancement in the rate of PET quenching of the fluorophore excited state from the anion:receptor moiety. This design can be further elaborated on by incorporating either two fluorophores, or urea/thiourea receptors into the sensor structures, using anthracene as a fluorophore. For the latter design, the sensors were designed to achieve sensing of bis-anions, such as di-carboxylates or pyrophosphate, where the anion bridged the anthracene moiety. In the case of the naphthalimide based mono-receptor based PET sensors, it was discovered that in DMSO the sensors were also susceptible to deprotonation by anions such as F− at high concentrations. This led to substantial changes in the absorption spectra of these sensors, where the solution changed colour from yellow/green to deep blue, which was clearly visible to the naked eye. Hence, some of the examples presented can act as dual fluorescent-colorimetric sensors for anions. Further investigations into this phenomenon led to the development of simple colorimetric sensors for fluorides, which upon exposure to air, were shown to fix carbon dioxide as bicarbonate.

Colorimetric and fluorescent anion sensors : an overview of recent developments in the use of 1,8-naphthalimide-based chemosensors

This critical review focuses on the development of anion sensors, being either fluorescent and/or colorimetric, based on the use of the 1,8-naphthalimide structure; a highly versatile building unit that absorbs and emits at long wavelengths. The review commences with a short description of the most commonly used design principles employed in chemosensors, followed by a discussion on the photophysical properties of the 4-amino-1,8-naphthalimide structure which has been most commonly employed in both cation and anion sensing to date. This is followed by a review of the current state of the art in naphthalimide-based anion sensing, where systems using ureas, thioureas and amides as hydrogen-bonding receptors, as well as charged receptors have been used for anion sensing in both organic and aqueous solutions, or within various polymeric networks, such as hydrogels. The review concludes with some current and future perspectives including the use of the naphthalimides for sensing small biomolecules, such as amino acids, as well as probes for incorporation and binding to proteins; and for the recognition/sensing of polyanions such as DNA, and their potential use as novel therapeutic and diagnostic agents (95 references).

Luminescent probes for the bioimaging of small anionic species in vitro and in vivo

The ability to spatiotemporally identify the formation of specific anionic species, or track changes in their concentration inside living systems, is of critical importance in deciphering their exact biological roles and effects. The development of probes (also called bioimaging agents and intracellular sensors) to achieve this goal has become a rapidly growing branch of supramolecular chemistry. In this critical review the challenges specific to the task are identified and for a select range of small anions of environmental and biological relevance (fluoride, chloride, iodide, cyanide, pyrophosphate, bicarbonate, hydrosulphide, peroxynitrite, hypochlorite and hypobromite) a comprehensive overview of the currently available in vitro and in vivo probes is provided.

Anion recognition and sensing in organic and aqueous media using luminescent and colorimetric sensors

This review article focuses primarily on the work carried in our laboratories over the last few years using luminescent and colorimetric sensors, where the anion recognition occurs through hydrogen bonding in organic or aqueous solvents. This review begins with the story of the discovery of fluorescent photoinduced electron transfer (PET) sensors for anions using charged neutral urea or thiourea receptors where both fluorescent and NMR spectroscopic methods monitored anion recognition. This work led to the development of dual luminescent and colorimetric anion sensors based on the use of the ICT based naphthalimide chromophore, where ions such as fluoride gave rise to changes in both the fluorescence and the absorption spectra of the sensors, but at different concentrations. Here, the former changes were due to hydrogen bonding interactions, whereas the latter was due to the deprotonation of acidic protons, giving rise to the formation of the bifluoride anion (HF2−). Modification of the 4-amino-l,8-naphthalimide moiety has facilitated the formation of colorimetric anion sensors that work both in organic or aqueous solutions. Such charge neutral receptor motifs have also been incorporated into organic scaffolds with norbomyl and calixarene backbones, which have enabled us to produce anion directed self-assembled structures.

Dual responsive chemosensors for anions : the combination of fluorescent PET (Photoinduced Electron Transfer) and colorimetric chemosensors in a single molecule

The design and synthesis of two novel fluorescent PET anion sensors is described, based on the principle of ‘fluorophore-spacer-(anion)receptor’. The sensors 1 and 2 employ simple diaromatic thioureas as anion receptors, and the fluorophore is a naphthalimide moiety that absorbs in the visible part of the spectrum and emits in the green. Upon recognition of anions such as F⁻ and AcO⁻ in DMSO, the fluorescence emission of 1 and 2 was ‘switched off’, with no significant changes in the UV–vis spectra. This recognition shows a 1:1 binding between the receptor and the anions. In the case of F⁻, further additions of the anion, gave rise to large changes in the UV–vis spectra, where the λ_max at 455 nm was shifted to 550 nm. These changes are thought to be due to the deprotonation of the 4-amino moiety of the naphthalimide fluorophore. This was in fact found to be the case, using simple naphthalimide derivatives such as 6. Sensors 1 and 2 can thus display dual sensing action; where at low concentrations, the fluorescence emission is quenched, and at higher concentrations the absorption spectra are modulated.