Fluorescence Experiments Research Articles

Iron Contamination in High-Enthalpy Test Facilities: OH PLIF Imaging Considerations

Thermometry measurements were performed in a scramjet combustor using thermally-assisted laser-induced fluorescence. Experimental data were obtained through laser-induced fluorescence where a laser beam was focused into the combustor and the OH () transition at 283.55 nm was excited. Measurements in the 360 nm spectral region resulted in detection of a strong fluorescence line. Investigation of this phenomenon led to the conclusion that when the OH line is chosen for excitation, another line of a different species that lies very close to is also unintentionally excited. The second species has been identified as iron, Fe I. The assumption is that the erosion of the shock tunnel walls introduced iron in the flow that is excited with the laser when exciting OH. Thus, it is very important to ensure that this is avoided in future. Instead, another OH excitation line should be used for OH excitation line in planar laser-induced fluorescence (PLIF) experiments where iron may potentially also be present in the flow. Alternatively, fluorescence from this iron line can be circumvented by proper filtering of the PLIF. A proposal is made to turn the iron contamination into advantage by using iron present in the non-combusting flow for PLIF.

Photosystem II core quenching in desiccated Leptolyngbya ohadii

Cyanobacteria living in the harsh environment of the desert have to protect themselves against high light intensity and prevent photodamage. These cyanobacteria are in a desiccated state during the largest part of the day when both temperature and light intensity are high. In the desiccated state, their photosynthetic activity is stopped, whereas upon rehydration the ability to perform photosynthesis is regained. Earlier reports indicate that light-induced excitations in Leptolyngbya ohadii are heavily quenched in the desiccated state, because of a loss of structural order of the light-harvesting phycobilisome structures (Bar Eyal et al. in Proc Natl Acad Sci 114:9481, 2017) and via the stably oxidized primary electron donor in photosystem I, namely P700+ (Bar Eyal et al. in Biochim Biophys Acta Bioenergy 1847:1267–1273, 2015). In this study, we use picosecond fluorescence experiments to demonstrate that a third protection mechanism exists, in which the core of photosystem II is quenched independently.

The application of bioactive pyrazolopyrimidine unit for the construction of fluorescent biomarkers

The involvement of biologically active nitrogen heterocycles in the construction of conjugated structures was important to improve the biocompatibility and optical properties of fluorescent probes. Herein, six pyrazolo [1, 5-a] pyrimidine derivatives (1a–f) were prepared by aldol or Suzuki reaction with the C-5 or C-7 sites of bioactive pyrazolo [1, 5-a] pyrimidine and different fluorophores. The optical properties in eight organic solvents were investigated; they showed excellent solvatochromism and the maximum emission wavelength range was from 443 nm to 678 nm. Besides, all of them exhibited high fluorescence quantum yields (0.44–0.98) in small polar solvents and they also exhibited excellent photostability. Subsequently, the fluorescent imaging experiments indicated that probes 1a–f could be used as lipid droplets (LDs) biomarkers for HeLa cells (cancer cells) and L929 cells (normal cells); moreover, the number of LDs in HeLa cells was found to be obviously more than in L929 cells, so probes 1a–f possessed great potential in distinguishing between normal cells and cancer cells, even in imaging and diagnosis of LDs-related diseases.

Design, assembly, characterization, and operation of double-stranded interlocked DNA nanostructures.

Mechanically interlocked DNA nanostructures are useful as flexible entities for operating DNA-based nanomachines. Interlocked structures made of double-stranded (ds) DNA components can be constructed by irreversibly threading them through one another to mechanically link them. The interlocked components thus remain bound to one another while still permitting large-amplitude motion about the mechanical bond. The construction of interlocked dsDNA architectures is challenging because it usually involves the synthesis and modification of small dsDNA nanocircles of various sizes, dependent on intrinsically curved DNA. Here we describe the design, generation, purification, and characterization of interlocked dsDNA structures such as catenanes, rotaxanes, and daisy-chain rotaxanes (DCRs). Their construction requires precise control of threading and hybridization of the interlocking components at each step during the assembly process. The protocol details the characterization of these nanostructures with gel electrophoresis and atomic force microscopy (AFM), including acquisition of high-resolution AFM images obtained in intermittent contact mode in liquid. Additional functionality can be conferred on the DNA architectures by incorporating proteins, molecular switches such as photo-switchable azobenzene derivatives, or fluorophores for studying their mechanical behavior by fluorescence quenching or fluorescent resonance energy transfer experiments. These modified interlocked DNA architectures provide access to more complex mechanical devices and nanomachines that can perform a variety of desired functions and operations. The assembly of catenanes can be completed in 2 d, and that of rotaxanes in 3 d. Addition of azobenzene functionality, fluorophores, anchor groups, or the site-specific linkage of proteins to the nanostructure can extend the time line.

Rare earth perovskite modified cobalt disulfide catalysts controlled by reaction solvent synthesis to form a p-n heterojunction

The effect of reaction solvent on the hydrogen evolution activity of novel CoS2/FeLaO3-4(CS/FLO-4) p-n heterojunction photocatalysts, synthesized using the solvothermal method, was assessed. The highest activity under visible light irradiation was recorded for the CS/FLO-4 catalyst prepared in ethanol. In fact, after 5 h of reaction, the H2-yield of the ethanol composite (279.7 μmol) was found to be 5.2 and 150.2 times greater than that of CoS2 microspheres and FeLaO3 nanoparticles, respectively. Time-resolved fluorescence (TRF) and photoelectrochemical experiments confirmed the effectiveness of space charge separation in the ethanol catalyst, whereas linear sweep voltammetry results showed that this material is characterized by high current response. The direction of electron migration in the p-n type heterojunction (p-FeLaO3/n-CoS2) was found to be the same as that of photoexcited electron transfer (from the conduction band of CoS2 to the valence band of FeLaO3), which enhances the photocatalytic hydrogen evolution activity of the composite material. This work provides a feasible and simple strategy for the design and synthesis of FeLaO3-doped composite catalysts with high photocatalytic activity.

Experimental M1 response of Ar40 as a benchmark for neutrino-nucleus scattering calculations

The excitation of atomic nuclei via magnetic dipole transitions is closely related to the inelastic neutral-current neutrino-nucleus (NC-$\ensuremath{\nu}\mathrm{A}$) scattering process due to the similarity of the transition operators. NC-$\ensuremath{\nu}\mathrm{A}$-scattering serves for the detection of supernova neutrinos and poses a significant source of background in modern liquid-argon based high-energy neutrino detection experiments. To enable tests of the reliability of predictions for neutrino-nucleus scattering, the magnetic dipole response of $^{40}\mathrm{Ar}$ below 7.7 MeV was characterized in a nuclear resonance fluorescence experiment using quasimonoenergetic $\ensuremath{\gamma}$-ray beams. The linear polarization of the beams allowed for assignments of electric or magnetic character to previously known dipole excitations. A total magnetic dipole strength of $0.{36}_{\ensuremath{-}0.05}^{+0.04}\phantom{\rule{4pt}{0ex}}{\ensuremath{\mu}}_{N}^{2}$ was identified in the energy range of the present experiment. Combined with data from previous measurements, the full magnetic dipole strength of $^{40}\mathrm{Ar}$ below the neutron separation threshold was investigated. Due to the low background in the energy range within the bandwidth of the $\ensuremath{\gamma}$-ray beams, the previous sensitivity limit was improved. A large-scale nuclear shell model calculation in the $sd\text{\ensuremath{-}}fp$ space satisfactorily agrees with the data in terms of excitation energies and strengths of the observed ${1}^{+}$ states.

Structural state recognition facilitates tip tracking of EB1 at growing microtubule ends

The microtubule binding protein EB1 specifically targets the growing ends of microtubules in cells, where EB1 facilitates the interactions of cellular proteins with microtubule plus-ends. Microtubule end targeting of EB1 has been attributed to high-affinity binding of EB1 to GTP-tubulin that is present at growing microtubule ends. However, our 3D single-molecule diffusion simulations predicted a ~ 6000% increase in EB1 arrivals to open, tapered microtubule tip structures relative to closed lattice conformations. Using quantitative fluorescence, single-molecule, and electron microscopy experiments, we found that the binding of EB1 onto opened, structurally disrupted microtubules was dramatically increased relative to closed, intact microtubules, regardless of hydrolysis state. Correspondingly, in cells, the blunting of growing microtubule plus-ends by Vinblastine was correlated with reduced EB1 targeting. Together, our results suggest that microtubule structural recognition, based on a fundamental diffusion-limited binding model, facilitates the tip tracking of EB1 at growing microtubule ends.

CAFE: a new, improved nonresonant laser-induced fluorescence instrument for airborne in situ measurement of formaldehyde

Abstract. NASA Compact Airborne Formaldehyde Experiment (CAFE) is a nonresonant laser-induced fluorescence instrument for airborne in situ measurement of formaldehyde (HCHO). The instrument is described here with highlighted improvements from the predecessor instrument, COmpact Formaldehyde FluorescencE Experiment (COFFEE). CAFE uses a 480 mW, 80 kHz laser at 355 nm to excite HCHO and detects the resulting fluorescence in the 420–550 nm range. The fluorescence is acquired at 5 ns resolution for 500 ns and the unique time profile of the HCHO fluorescence provides measurement selectivity. CAFE achieves a 1σ precision of 160 pptv (1 s) and 90 pptv (10 s) for [HCHO] = 0 pptv. The accuracy of CAFE, using its curve-fitting data processing, is estimated as ±20 % of [HCHO] + 100 pptv. CAFE has successfully flown on multiple aircraft platforms and is particularly well-suited to high-altitude research aircraft or small air quality research aircraft where high sensitivity is required but operator interaction and instrument payload is limited.

Capturing the Quenching Mechanism of Light-Harvesting Complexes of Plants by Zooming in on the Ensemble

Summary The light-harvesting complexes (LHCs) of plants can regulate the energy flux to the reaction centers in response to fluctuating light by virtue of their vast conformational landscape. They do so by switching from a light-harvesting state to a quenched state, dissipating the excess absorbed energy as heat. However, isolated LHCs are prevalently in their light-harvesting state, which makes the identification of their photoprotective mechanism extremely challenging. Here, ensemble time-resolved fluorescence experiments show that monomeric CP29, a minor LHC of plants, exists in various emissive states with identical spectra but different lifetimes. The photoprotective mechanism active in a subpopulation of strongly quenched complexes is further investigated via ultrafast transient absorption spectroscopy, kinetic modeling, and mutational analysis. We demonstrate that the observed quenching is due to excitation energy transfer from chlorophylls to a dark state of the centrally bound lutein.

Solvation properties of raft-like model membranes

Dimethyl sulfoxide (DMSO) is a universal water-soluble solvent widely used in many biotechnological and medical applications, such as cells cryopreservation, and for the treatment of different human diseases (e.g. amyloidosis). Despite the great number of reported studies, the effects of DMSO on the physico-chemical properties of biological membranes are poorly understood. Often, these studies are limited to model membranes composed of phosphatidylcholines (PCs) and cholesterol (Chol). In this work, we explored the effect of DMSO on liposomes composed of the natural egg sphingomyelin (ESM) and Chol as raft-like model membranes.With a multi-technique approach we probe the structure and the thermal stability of ESM/Chol bilayer at different Chol mole fractions. In particular, we investigate the ESM-solvent interactions to clarify the role of DMSO in perturbing the solvating conditions of lipid vesicles and show that the addition of DMSO increases the thermal stability of vesicles. An increase of transition temperature, a decrease of both enthalpy and entropy as well as a decrease of the cooperativity of the gel to liquid phase transition are observed at 0.1 DMSO mole fraction. Fluorescence experiments with the probe Laurdan and FTIR spectra strongly indicate that DMSO exerts a dehydration effect on the membrane. Besides, FTIR measurements with tungsten hexacarbonyl, in combination with fluorescence data of the probe NBD-PE, indicate that DMSO promotes the formation of a highly packed membrane by reducing the thickness of the membrane.

Epitope Mapping by NMR of a Novel Anti-A\u03b2 Antibody (STAB-MAb)

Alzheimer´s Disease (AD) is one of the most common neurodegenerative disorders worldwide. Excess of β-amyloid (Aβ), a peptide with a high propensity to misfold and self-aggregate, is believed to be the major contributor to the observed neuronal degeneration and cognitive decline in AD. Here, we characterize the epitope of a novel anti-Aβ monoclonal antibody, the STAB-MAb, which has previously demonstrated picomolar affinities for both monomers (KD = 80 pM) and fibrils (KD = 130 pM) of Aβ(1–42) and has shown therapeutic efficacy in preclinical mouse models of AD. Our findings reveal a widespread epitope that embraces several key Aβ residues that have been previously described as important in the Aβ fibrillation process. Of note, STAB-MAb exhibits a stronger affinity for the N-terminus of Aβ and stabilizes an α-helix conformation in the central to N-terminal region of the peptide, in addition to disrupting a characteristic salt-bridge of a hairpin structure present in fibrils. The NMR derived epitope supports the observed results from ThT-monitored fluorescence and electron microscopy experiments, in which STAB-MAb was shown to inhibit the formation of aggregates and promote disruption of pre-formed fibrils. In combination with the published in vitro and in vivo assays, our study highlights STAB-MAb as a rare and versatile antibody with analytical, diagnostic and therapeutic efficacy.

Functional Characterization of a Drought-Responsive Invertase Inhibitor from Maize (Zea mays L.).

Invertases (INVs) play essential roles in plant growth in response to environmental cues. Previous work showed that plant invertases can be post-translationally regulated by small protein inhibitors (INVINHs). Here, this study characterizes a proteinaceous inhibitor of INVs in maize (Zm-INVINH4). A functional analysis of the recombinant Zm-INVINH4 protein revealed that it inhibited both cell wall and vacuolar invertase activities from maize leaves. A Zm-INVINH4::green fluorescent protein fusion experiment indicated that this protein localized in the apoplast. Transcript analysis showed that Zm-INVINH4 is specifically expressed in maize sink tissues, such as the base part of the leaves and young kernels. Moreover, drought stress perturbation significantly induced Zm-INVINH4 expression, which was accompanied with a decrease of cell wall invertase (CWI) activities and an increase of sucrose accumulation in both base parts of the leaves 2 to 7 days after pollinated kernels. In summary, the results support the hypothesis that INV-related sink growth in response to drought treatment is (partially) caused by a silencing of INV activity via drought-induced induction of Zm-INVINH4 protein.

Integrated system for temperature-controlled fast protein liquid chromatography. III. Continuous downstream processing of monoclonal antibodies

Three different applications of travelling heating zone reactor (THZR) chromatography for the downstream processing of monoclonal antibodies (mAbs) are described. mAb containing feedstocks were applied to a fixed bed of the thermoresponsive rProtein A matrix, Byzen Pro™, contained in a bespoke column (held at 15 °C) fitted with a travelling heating (42 °C) device encircling a narrow section of the column. For the demonstration of continuous concentration, uninterrupted loading of 1.0 g/L mAb in a pH 8 binding buffer was synchronized with 5 repeated movements of the heating zone along the column’s full length at a velocity of 0.1 mm/s. Elution of mAbs was induced solely by the travelling heating zone’s action, each full movement generating a sharp concentrated elution peak accompanied by a small transient mAb concentration-dependent dip in conductivity. Quasi-steady-state operation occurred from the third elution onwards, delivering a mean mAb concentration of 4.9 g/L and process yield >93%. Quasi-continuous separation of the target mAb (1.41 g/L) from bovine serum albumin, BSA (1.0 g/L), was achieved by cyclically alternating the feeding of the mAb + BSA feedstock, with that of the binding buffer alone; supply of the latter was timed to coincide with movement of the heating zone. Accurate coordination of the heating zone’s travel and switching from feed to buffer permitted quasi-steady-state collection (elutions 3–6) of sharp peaks of mAb in high purity (98.7%) and yield (88.7%) in 4.5–fold concentrated form, with BSA exiting in the flow through fractions between successive mAb elution peaks. Fully automated THZR-mediated quasi-continuous buffer exchange of 1.34 g/L mAb from a phosphate buffer pH 8 into a HEPES buffer pH 8 of slightly lower conductivity was performed over a 19 h period by carefully timed switching from one feed solution to the other and back again, whilst synchronising movement of the heating zone with feeding of the exchange buffer. Quasi-steady-state operation (elutions 2–9) resulted in an average eluted mAb yield of 94.5% and concentration of 4.8 g/L. Triggering movement of the heating zone slightly ahead of the switch from mAb feed to exchange buffer permitted the positioning of mAb elution peaks in 9 mL volume segments with the lowest recorded conductivity. Measurements of buffer exchange performance conducted with two ‘protein-free’ systems demonstrated that compared to tangential flow filtration in diafiltration mode, which represents the ‘state-of-the-art’ technology for buffer exchange, the THZR chromatography based approach affords a >60% saving in minimum volume of exchange buffer required to remove 99.9% of the original buffer. Combined far and near UV circular dichroism, intrinsic fluorescence and thermal melting experiments showed that, unlike conventional Protein A/G affinity chromatography, the conditions for THZR Protein A chromatography respect maintenance of a favourable structural profile for mAbs.

A multifunctional lipid that forms contrast-agent liposomes with dual-control release capabilities for precise MRI-guided drug delivery

Monitoring of nanoparticle-based therapy in vivo and controlled drug release are urgently needed for the precise treatment of disease. We have synthesized a multifunctional Gd-DTPA-ONB (GDO) lipid by introducing the Gd-DTPA contrast agent moiety into an o-nitro-benzyl ester lipid. By design, liposomes formed from the GDO lipid combine MRI tracking ability and dual-trigger release capabilities with maximum sensitivity (because all lipids bear the cleavable moiety) without reducing the drug encapsulation rate. We first confirmed that both photo-treatment and pH-triggered hydrolysis are able to cleave the GDO lipid and lyse GDO liposomes. We then investigated the efficiency of drug release via the combined release processes for GDO liposomes loaded with doxorubicin (DOX). Relative to neutral pH, the release efficiency in acidic environment increased by 10.4% (at pH = 6.5) and 13.3% (at pH = 4.2). This pH-dependent release response is conducive to distinguishing pathological tissue such as tumors and endolysosomal compartments. The photo-induced release efficiency increases with illumination time as well as with distance of the pH from neutral. Photolysis increased the release efficiency by 13.8% at pH = 4.2, which is remarkable considering the already increased amount of drug release in the acidic environment. In addition, the relaxation time of GDO liposomes was 4.1 times that of clinical Gd-DTPA, with brighter T1-weighted imaging in vitro and in vivo. Real-time MRI imaging and in vivo fluorescence experiments demonstrated tumor targeting and MRI guided release. Furthermore, significant tumor growth inhibition in a treatment experiment using DOX-loaded GDO liposomes clearly demonstrated the benefit of photo-treatment for efficacy: the tumor size in the photo-treatment group was 3.7 times smaller than in the control group. The present study thus highlights the benefit of the design idea of combining efficient imaging/guiding, targeting, and triggerable release functions in one lipid molecule for drug delivery applications.

Direct measurement of deubiquitinating enzyme activity in intact cells using a protease-resistant, cell-permeable, peptide-based reporter

Deubiquitinating enzymes (DUBs) regulate the removal of the polyubiquitin chain from proteins targeted for degradation. Current approaches to quantify DUB activity are limited to test tube-based assays that incorporate enzymes or cell lysates, but not intact cells. The goal of this work was to develop a novel peptide-based biosensor of DUB activity that is cell permeable, protease-resilient, fluorescent, and specific to DUBs. The biosensor consists of an N-terminal β-hairpin motif that acts as both a ‘protectide’ to increase intracellular stability and a cell penetrating peptide (CPP) to facilitate the uptake into intact cells. The β-hairpin was conjugated to a C-terminal substrate consisting of the last four amino acids in ubiquitin (LRGG) to facilitate DUB mediated cleavage of a C-terminal fluorophore (AFC). The kinetics of the peptide reporter were characterized in cell lysates by dose response and inhibition enzymology studies. Inhibition studies with an established DUB inhibitor (PR-619) confirmed the specificity of both reporters to DUBs. Fluorometry and fluorescent microscopy experiments followed by mathematical modeling established the capability of the biosensor to measure DUB activity in intact cells while maintaining cellular integrity. The novel reporter introduced here is compatible with high-throughput single cell analysis platforms such as FACS and droplet microfluidics facilitating direct quantification of DUB activity in single intact cells with direct application in point-of-care cancer diagnostics and drug discovery.

Probing the interaction of carbonaceous dots with transferrin and albumin: Impact on the protein structure and non-synergetic metal release

Carbon quantum dots (CQD) have been emerging as an essential material for biological applications due to their relevant properties. Here, we synthesized and characterized low toxic anionic carbon dots and examined the interaction of the nanoparticles with human transferrin (hTf) and bovine serum albumin (BSA) by using multi-spectroscopic techniques. CQD showed low cytotoxic effect against HBMEC, A549, and HTC116 cells. Temperature variable fluorescence experiments reveal that CQD quench the BSA and hTf intrinsic fluorescence following a dynamical and static mechanisms, respectively. Both proteins attach tightly to the nanoparticle (Ka ≈ 104–105 M−1), and hydrophobic forces drive the binding. Additionally, hTf-CQD interaction is also guided by electrostatic interaction. Furthermore, protein CD spectra reveal that both proteins experience structural changes during nanoparticle interaction, which differs from previously reported anionic carbon dots from other sources. Experiments corroborate the observed structural modifications and unveil that the nanoparticle interaction induces iron release from hTf lobes. The mechanism of hTf iron release induced by CQD can be understood based on anionic non-synergetic effects.

Development of a two-photon carbazole derivative probe for fluorescent visualization of G-quadruplex DNA in cells

G-quadruplex (G4) sequences are considered to play important roles in gene regulation, therefore the development of selective and sensitive probes for G4 DNAs is important for studying the functions of G-rich gene sequences, as well as designing of novel and effective anticancer drugs. Herein, a carbazole derivative (CZ-BT) was synthesized and characterized by a simple process for G4 DNA detection. CZ-BT was preferentially bound with G4 DNA compared with other types of nucleic acids according to the fluorescence assays. The fluorescence intensity and fluorescence lifetime of CZ-BT significantly increased after the interaction with G4 DNA. Molecular docking calculation proved the π-π stacking binding mode between CZ-BT and G4 DNA. Furthermore, the specificity of CZ-BT on G4 DNA was well demonstrated with the contrast of pyridostatin (PDS, a classical G-quadruplex ligand) using two-photon confocal fluorescent imaging technique and cell cycle experiment in cells. We believe that this study would give some favorable factors on developing of highly effective fluorescent probes for G4 DNA applications.

A novel fluorescent probe based on isocoumarin for Hg2+ and Fe3+ ions and its application in live-cell imaging.

Synthesis of the 2-amino-4-phenyl-6- (isocoumarin-3-yl) -3-cyanopyridine (APICP) containing both isocoumarin and pyridine ring in its structure was carried out, and this compound was characterized by ATR-FTIR, 1H NMR, and 13C NMR spectral techniques. A fluorescence sensor determining Hg2+ and Fe3+ ions in DMSO/HEPES buffer solution (9/1 v/v, 5 μM, pH 7.0) was developed using the synthesized compound, and the detection limits of the sensor with exquisite selectivity were calculated as 8.12 nM and 5.51 nM for Hg2+ and Fe3+ ions, respectively. Jobs plot method was used to determine the stoichiometry of APICP-Hg2+/Fe3+ complexes as 2:1 and FT-IR and ESI-MS methods confirmed the results. Besides, cell growth inhibitory potentials of the sensor over HepG2 cells and invivo fluorescent cell imaging experiments were conducted. Findings revealed the relatively low cytotoxic effects of the synthesized sensor (IC50: 0.541 ± 0.039 mM), and it could be utilized as an intracellular imaging agent for the determination of Fe3+ and Hg2+ ions in biological systems.

An experimental and computational study on naphthylideneimine based pH sensitive fluorescence probe for zinc.

Rational design chelating fluorescent sensors probing metal ions in biological system are continuously hot essays nowadays, especially for zinc detection. Herein, a naphthylideneimine based zinc fluorescence probe (3) was prepared and characterized in this work. Structural features and optical properties of 3 and its metallic complexes were characterized. Fluorescent experiment indicates 3 is extremely sensitive and selective for Zn2+ with a strong fluorescence enhancement (∼34 folds) in aqueous buffer solution with a limit of detection (LOD) of 3.78 × 10-7 mol L-1. Formation constant (logKa) of the chelating complex of 3 and Zn2+ ion was determined to be 4.45. Theoretical studies were carried out to get deep insight into the response mechanism in the sensing process. Density functional theory (DFT) methods calculated formation Gibbs free energy (ΔrGmө) of the deprotonated complexes model (32- ⊃ Zn) is-2.9 kcal/mol, which is in good agreement with the experimental result. The calculation results show that the low excitation states can be ascribe to S0 → T2 and S0 → S1 at 390-430 nm and 310-330 nm, respectively, due to the π → π∗ transition. Finally, yeast cell imaging experiments indicate that 3 can monitor intracellular Zn2+ as well. These findings would enable this fluorescent probe to be used as a Zinc sensor.

Screening and characterization of a novel high-efficiency tumor-homing cell-penetrating peptide from the buffalo cathelicidin family.

Targeted delivery of antitumor drugs is especially important for tumor therapy. Cell-penetrating peptides (CPPs) have been shown to be very effective drug carriers for tumor therapy. However, most CPPs lack tumor cell specificity. Here, we identified a highly efficient CPP, CAT, from the newly identified buffalo-derived cathelicidin family, which exhibits a preferential binding capacity for multiple tumor cell lines and delivers carried drug molecules into cells. CAT showed an approximately threefold to sixfold higher translocation efficiency than some reported cell-penetrating antimicrobial peptides, including the well-known classical CPP TAT. Moreover, the delivery efficiency of CAT was greater in a variety of tested tumor cells than in normal cells, especially for the human hepatoma cell line SMMC-7721, for which delivery was 7 times more efficient than the normal human embryonic lung cell line MRC-5, according to fluorescent labeling experiment results. CAT was conjugated to the Momordica charantia-derived type-I ribosome-inactivating protein MAP 30, and the cytotoxicity of the MAP 30-CAT fusion protein in the tumor cell line SMMC-7721 was significantly enhanced compared with that of the unconjugated MAP 30. The IC50 value of MAP 30-CAT was approximately 83 times lower than the IC50 value of the original MAP 30. Interestingly, the IC50 value of MAP 30 alone for MRC-5 was approximately twofold higher than the value for SMMC-7721, showing a small difference. However, when MAP 30 was conjugated to CAT, the difference in IC50 values between the two cell lines was significantly increased by 38-fold. The results of the flow cytometric detection of apoptosis revealed that the increase in cytotoxicity after CAT conjugation was mainly caused by the increased induction of apoptosis by the fusion protein. These results suggest that CAT, as a novel tumor-homing CPP, has great potential in drug delivery applications in vivo and will be beneficial to the development of tumor therapeutics.