Fluorescence Experiments Research Articles

Synthesis and in vitro evaluation of chlorogenic acid amides as potential hypoglycemic agents and their synergistic effect with acarbose

Type 2 Diabetes mellitus is a chronic disease considered one of the most severe global health emergencies. Chlorogenic acid (1) has been shown to delay intestinal glucose absorption by inhibiting the activity of α-glucosidase (α-Glu) and α-amylase (α-Amy). In the present work, eleven chlorogenic acid amides have been synthesized and evaluated for their antioxidant properties (as DPPH and ORAC) and inhibition activity towards the two enzymes and, with the aim to obtain dual-action antidiabetic agents. The two most promising hypoglycemic compounds, bearing a tertiary amine function on an alkyl chain (8) and a benzothiazole scaffold (11), showed IC50 values lower than that of (1) (45.5 µM α-Glu; 105.2 µM α-Amy). Amides 8 and 11 were by far more potent α-Glu inhibitors than the antidiabetic drug acarbose (IC50 = 268.4 µM) and about twice less active toward α-Amy than acarbose (IC50 = 34.4 µM). Kinetics experiments on amides 8 and 11 indicated these compounds as mixed-type inhibitors of α-Glu with Kʹi values of 13.3 and 6.3 µM, respectively. The amylase inhibition occurred with a competitive mechanism in the presence of 8 (Ki = 79.7 µM) and with a mixed-type mechanism with 11 (Ki = 19.1 µM; Kʹi = 93.6 µM). Molecular docking analyses supported these results, highlighting the presence of additional binding sites in both enzymes. Fluorescence experiments confirmed the grater affinity of amides 8 and 11 towards the two enzymes respect to (1). Moreover, a significant enhancement in acarbose efficacy was observed when inhibition assays were performed adding acarbose and amide 11. The above outcomes pinpointed the benzothiazole-based amide 11 as a promising candidate for further studies on type 2 diabetes treatment, both alone or combined with acarbose.

Zwitterionic daptomycin stabilized palladium nanoparticles with enhanced peroxidase-like properties for glucose detection

Mimic enzymes, especially noble metal nanomaterials, replace natural horseradish peroxidase due to the artificial regulation of catalytic activity by adjusting particle size and morphology. However, the existing mimic enzymes still have disadvantages such as large particle size, easy clustering and poor biocompatibility. Herein, well-dispersed daptomycin stabilized palladium nanoparticles (Dap-Pdn NPs) were synthesized by using daptomycin as a biological template. The average particle size of palladium nanoparticles of Dap-Pd0.25 NPs was 1.73 nm, the hydrodynamic size of Dap-Pd0.25 NPs detected by DLS method was 49.97 nm, Dap-Pd0.25 NPs were stable in fibrinogen solution. In addition, Dap-Pd0.25 NPs had excellent peroxidase-like activity, which effectively catalyzed the colorimetric oxidation reaction of 3,3′,5,5′-tetramethylbenzidine (TMB) by H2O2. Dap-Pd0.25 NPs were used to detect glucose concentration based on the colorimetric reaction of TMB, which had a wide detection range (0.02–1 mM) and low detection limit (5.6 μM) for glucose. Besides, fluorescence experiments proved that Dap-Pdn NPs catalyzed H2O2 to generate·OH. Taken together, a simple, green and inexpensive synthesis method of palladium nanoparticles with enhanced peroxidase-like activity was proved in this research. Dap-Pdn NPs will have great application prospects in biological detection in the future.

Chirally modified cobalt-vanadate grafted on battery waste derived layered reduced graphene oxide for enantioselective photooxidation of 2-naphthol: Asymmetric induction through non-covalent interaction

The cobalt oxide-vanadium oxide (Co3O4-V2O5) combined with reduced graphene oxide (rGO) having band gap of ∼ 3.3 eV appeared as a suitable photocatalyst for selective oxidation of 2-naphthol to BINOL. C2-symmetric BINOL was achieved with good yield using hydrogen peroxide as the oxidant under UV-light irradiation. The same catalyst was chirally modified with cinchonidine and a newly synthesized chiral Schiff base ligand having a sigma-hole center. The strong interaction of the chiral modifiers with the cobalt-vanadium oxide was truly evident from various spectroscopic studies and DFT calculations. The chirally modified mixed metal oxide transformed the oxidative CC coupling reaction with high enantioselectivity. High enantiomeric excess upto 92 % of R-BINOL was obtained in acetonitrile solvent and hydrogen peroxide as the oxidant. A significant achievement was the formation of S-BINOL in the case of the cinchonidine modified catalyst and R-BINOL with the Schiff base ligand anchored chiral catalyst. The UV-light induced catalytic reaction was found to involve hydroxyl radical as the active reactive species. The spin trapping ESR and fluorescence experiment provided relevant evidence for the formation of such species through photodecomposition of hydrogen peroxide on the catalyst surface. The chiral induction to the resultant product was found to induce through supramolecular interaction like OH…π, H…Br interaction. The presence of sigma hole center was believed to play significant role in naphtholate ion recognition during the catalytic cycle.

A Cu(I) complex groove binder with a high affinity towards DNA denaturation

The interaction of ligands with double-stranded DNA is important for many intracellular processes. We demonstrate the ability of a copper(I) bis-triphenylphosphine based complex with a high affinity for target DNA via a minor groove binding mode. The fluorescence and UV–visible results show that a complex-DNA conjugate forms in the ground state with a binding affinity in order of 10+5 M−1. According to the findings of competitive fluorescence experiments, the phosphine substituent attaches in the minor groove of DNA by replacing Hoechst molecules at A-T rich regions. Also, relatively small changes in the CD spectrum of DNA as well as in its viscosity indicated that the Cu(I) complex could interact as a groove binder. The resulted entropy and enthalpy changes denoting the Van der Waals forces as the key binding mechanism in the interaction. Fluorescent DNA melting analysis shows that the Cu(I) complex is strongly disturbed the stability of the DNA base pairs, making the dsDNA to ssDNA easier to transition. The insertion of phosphine bulky substituents in the DNA minor groove was approved by docking analysis. Moreover, the molecular dynamic simulation shows an opening of DNA chains due to the Cu(I) complex force applied.

Deciphering the biomolecular interaction of β-resorcylic acid with human lysozyme: A biophysical and bioinformatics outlook

The binding mechanism between human lysozyme (Lyz) and β-resorcylic acid (BR) was systematically examined by various biophysical and computational techniques. The studies like ultraviolet absorbance and steady-state fluorescence confirmed the complex formation with binding constant in the order of 103 M−1. The mechanism of fluorescence quenching of Lyz was found to be static. The average binding distance between Lyz and BR was calculated by FRET as per Forster’s theory. The changes in conformation and microenvironment in the secondary structure of Lyz in the presence of BR were confirmed by techniques like synchronous, three-dimensional fluorescence spectroscopy, Red edge excitation shift (REES) and circular dichroism (CD). The different temperature-based fluorescence experiments highlighted that the reaction is spontaneous and exothermic in nature. The van der Waals and hydrogen bonding are the main contributing forces in the complex formation as is evident from the negative values of enthalpy (ΔH) and entropy (ΔS). Further, the results obtained from in vitro experiments were also reaffirmed by in silico molecular docking, predicting the active binding site of BR in Lyz. HOMO-LUMO energy gap in BR was estimated by density functional theory (DFT) studies. Moreover, the effects of β-cyclodextrin, metal ions, and vitamins on the binding of BR with Lyz were also studied.

Free Radical Generation in Far-UV Synchrotron Radiation Circular Dichroism Assays-Protein and Buffer Composition Contribution.

A useful tool to analyze the ligands and/or environmental contribution to protein stability is represented by the Synchrotron Radiation Circular Dichroism UV-denaturation assay that consists in the acquisition of several consecutive repeated far-UV SRCD spectra. Recently we demonstrated that the prevailing mechanism of this denaturation involves the generation of free radicals and reactive oxygen species (ROS). In this work, we analyzed the effect of buffering agents commonly used in spectroscopic measurements, including MOPS (3-(N-morpholino) propanesulfonic acid), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TRIS-HCl (tris-hydroxymethil aminomethane hydrochloride), and phosphate, on the efficiency of protein denaturation caused by exposure to UV radiation. Fluorescence experiments confirmed the presence of ROS and were used to determine the rate of ROS generation. Our results indicate that the efficiency of the denaturation process is strongly influenced by the buffer composition with MOPS and HEPES acting also as scavengers and that the presence of proteins itself influenced the ROS formation rate.

Comparative scrutinize of BSA and HEWL in the vicinity of metallo-catanionic aggregates derived from single chain metallosurfactant and anionic surfactant

In recent times, protein-surfactant interactions have been extensively explored due to their stimulating applications in different fields. These systems have efficaciously addressed the economic, environmental, and ecological issues. Sensing their prominence, through this report, we investigate and compare the interactional behavior of Bovine Serum Albumin (BSA) and Hen Egg White Lysozyme (HEWL) with metallo-catanionic aggregate mixtures. Attempts have been made to synthesize the metallo-catanionic aggregates from single-chain metallosurfactant (FeCTAC I) and anionic surfactant (SDS) i.e., FeCTAC I:SDS. Before proceeding towards the interactional studies of metallo-catanionic aggregates with model proteins, the morphology of formed aggregates was elucidated. Here, out of different possible molar ratios of FeCTAC I:SDS, two ratios i.e 70:30 and 30:70 were opted. The aggregates were examined through conductivity, Dynamic Light Scattering (DLS), Field Emission Scanning Electron Microscopy (FESEM), Confocal Laser Scanning Microscopy (CLSM), Small Angle X-Ray Scattering (SAXS), Zeta Potential, and Vibrating Sample Magnetometer (VSM) techniques. Interestingly, the SAXS analysis demonstrated the micellar behavior of 70:30 and vesicular behavior of 30:70 aggregates mixture, due to the difference of overall charge on them. The interactional study of BSA and HEWL with FeCTAC I:SDS was carried out through SDS-PAGE, absorption, emission, and circular dichroism (CD) spectroscopy. The effect of FeCTAC I:SDS on the linear polypeptide chain of BSA and HEWL was observed to be different, however, the secondary and tertiary structures were affected similarly. To elucidate the exact locality of FeCTAC I:SDS aggregates on the BSA, a site marker experiment was carried out. The pyrene extrinsic fluorescence experiment was performed to verify the type of interactions among HEWL and metallo-catanionic aggregates. From, the comparative studies, we concluded that the overall system was controlled by the type of forces playing between the protein and aggregate mixture that emerged from the total charge on the protein as well as on the aggregates. These types of metallo-catanionic aggregates that are flexible to shape are considered as the new alternatives for nano-vehicle applications.

Precise Detection and Visualization of Cyclooxygenase-2 for Golgi Imaging by a Light-Up Aggregation-Induced Emission-Based Probe

Precise Detection and Visualization of Cyclooxygenase-2 for Golgi Imaging by a Light-Up Aggregation-Induced Emission-Based Probe

Multifarious Impact of Rhodanine Acceptor Group on the Optical Properties of Some Semiconductor Probes

AbstractSteady state optical properties such as photo‐absorption and photo‐luminescence characteristics of organic molecules in its bulk states viz, film or crystalline forms are highly sensitive to subtle alteration of chemical substitution, molecular geometry in bulk and the electronic state coupling between a pair of molecules. Here we highlight a distinct interference of rhodanine substitution on such optical properties of a group of similar molecules (2TR, 23TR). For comparison an anthracene conjugate model molecule was also chosen (AR). In the former case, despite of having similar molecular structure except the presence of a methyl group at the C3 position (which ensures the identical electronic states in both the case) and similar molecular packing in single crystals, 2TR and 23TR exhibited diagonally opposite solid‐state fluorescence behavior. 2TR was non‐fluorescent whereas, 23TR was highly emissive in its crystalline solid state. A minute substitutional change in chemical structure was found to alter the solid‐state fluorescence behavior immensely that was attributed to the non‐radiative loss in the former case due to the solid‐state C−C bond flip which was further proven by temperature dependent fluorescence experiments. At a sample temperature of <120 K, 20‐fold enhancement in the fluorescence intensity was observed.

MOF-derived magnetically recoverable Z-scheme ZnFe2O4/Fe2O3 perforated nanotube for efficient photocatalytic ciprofloxacin removal

Constructing advanced direct Z-scheme photocatalysts is an effective strategy to enhance photogenerated charge separation for photocatalytic removal of organic pollutants but faces challenges in efficient morphology control and recovery method. Herein, a novel magnetically recoverable Z-scheme ZnFe2O4/Fe2O3 perforated nanotube was successfully prepared by one-step MOF-derived calcination method by using MIL-88B/Zn core/shell nanorod as precursor for the first time. In the calcination process, MIL-88B core reacted with oxygen to form hollow Fe2O3 and Zn2+ shell underwent an in situ solid-state reaction with Fe2O3 to cause volume shrinkage and formation of ZnFe2O4, and finally turned into intimate ZnFe2O4/Fe2O3 perforated nanotube (ZFF). Because of such exquisitely perforated nanotube and Z-scheme transfer pathway, ZFF shows superior light absorption, bulk-phase and interfacial separation of photogenerated charge, and thus lead to prominent photocatalytic performance for ciprofloxacin (CIP) removal. Especially, ZFF-2 exhibits the best photocatalytic CIP degradation performance with a degradation percentage of 96.5% and a TOC removal percentage up to 89% under light irradiation for 180 min. Moreover, ZFF show sufficient photo-stability and re-usability for photocatalytic degradation of CIP by a convenient magnetic recovery method. The electron spin resonance experiments, radical trapping and fluorescence experiments reveal that the photogenerated charge pathways of ZFF photocatalysts is a Z-scheme mechanism and the major active species for degradation of CIP are •O2– and •OH. This work offers a new perspective on construction of high-efficient direct Z-scheme photocatalysts with convenient recovery and deep insight on pollutants purification mechanism.

Design, Synthesis, and Herbicidal Activity of Thioether Containing 1,2,4-Triazole Schiff Bases as Transketolase Inhibitors

Transketolase (TK) represents a potential target for novel herbicide development. To discover novel TK inhibitors with potency against resistant weeds, 36 thioether compounds containing 1,2,4-triazole Schiff bases were designed and synthesized for herbicidal activity evaluation. The results demonstrated that compounds 5av and 5aw provided excellent weed control with inhibition of over 90% against the tested weeds, even at concentrations as low as 100 mg/L in vitro. In addition, compounds 5av and 5aw exhibited higher postemergence herbicidal activity than all of the positive controls against the tested weeds at 50-90 g [active ingredient (ai)]/ha in a greenhouse, while being safe for crops of maize and wheat at 90 g (ai)/ha. Fluorescent binding experiments of At TK indicated that compounds 5av and 5aw had strong TK inhibitory activity and could tightly bind with the enzyme At TK. Also, molecular docking analyses revealed that the structures of compounds 5av and 5aw were suitable for TK inhibitory activity. Taken together, these results suggested that compounds 5av and 5aw were promising herbicide candidates for weed control in wheat and maize fields targeting TK.

Engineering of anticancer drugs entrapped polymeric nanoparticles for the treatment of colorectal cancer therapy

Traditional chemotherapeutic agents have been successfully used to treat cancers, although their many disadvantages include poor solubility, high dosage needs, severe side effects, low therapeutic indices, development of multiple drug resistance, and non-specific targeting of cancer cells. In this study, Methotrexate (MEX) and curcumin (CUR) were incorporated into the triblock copolymer poly(lactic-co-glycolic acid) (PLGA)–poly(ethylene glycol) (PEG)–PLGA to achieve synergism in the treatment of colorectal cancer. Nanoparticle-based combination treatments for colorectal cancer can be developed using this remarkably symmetric synthetic strategy. The particles were visualized by high-resolution transmission microscopy (HR-TEM), and their size and polydispersity were determined using dynamic light scattering. Without a blast of impact, MEX and CUR were released gently from the polymeric nanoparticle system. CUR/MEX@NPs also demonstrated dose-dependent cytotoxic effects on SW1417 and CL40 cell lines, with a higher apoptosis ratio than free MEX and CUR. According to the in vitro cellular uptake analysis, cancer cells absorbed the polymeric nanoparticles more readily. From the fluorescence experiment, nanomaterials promoted cell death in the SW1417 and CL40 cell lines. Additionally, in vivo systemic toxicity and in vitro findings support the biocompatibility of CUR/MEX@NPs. Furthermore, these nanomaterials cause apoptosis in cancer cells. From these findings, CUR/MEX@NPs exhibit the potential to be successfully utilized to treat colorectal cancer.

Quest for the quenching and binding mode of functionalized ZnO QDs with calf thymus DNA: Biophysical and in silico molecular modelling approach

The effect of capping on zinc oxide (ZnO) quantum dots (QD)upon its interaction with calf thymus DNA (ct-DNA) in vitro have been examined using different specrtroscopic techniques. The experimental results elucidate two possible binding modes between ZnO QDs and ct-DNA. The quenching mechanism of QDs in presence of DNA revealed that although two types of quenching is possible but overall quenching is dominated by complex formation (i.e. static quenching) over dynamic collision (i.e. dynamic quenching). In our steady state absorption and fluorescence experiments the binding constant came up in the order of 104 L mol−1 which is an indicator of intercalation. The competitive displacement assay, CD spectral analysis, iodide quenching further reflect threaded intercalation between QDs and ct-DNA. Here, most of the QDs may bind to the DNA as groove binder and the rest as intercalator. Time-resolved measurements and in silico experiments also confirmed our conclusion. Usually, the size of QDs compels itself to bind as groove binder with ct-DNA, but in our study, we have detected that after suitable capping QD can behave as a mixed binder with ct-DNA. As QDs are frequently used as a drug carrier, this study will help in drug designing research.

An Open-Source Framework for Automated High-Throughput Cell Biology Experiments.

Modern data analysis methods, such as optimization algorithms or deep learning have been successfully applied to a number of biotechnological and medical questions. For these methods to be efficient, a large number of high-quality and reproducible experiments needs to be conducted, requiring a high degree of automation. Here, we present an open-source hardware and low-cost framework that allows for automatic high-throughput generation of large amounts of cell biology data. Our design consists of an epifluorescent microscope with automated XY stage for moving a multiwell plate containing cells and a perfusion manifold allowing programmed application of up to eight different solutions. Our system is very flexible and can be adapted easily for individual experimental needs. To demonstrate the utility of the system, we have used it to perform high-throughput Ca2+ imaging and large-scale fluorescent labeling experiments.

Adhesion behavior of silica nanoparticles with bacteria: Spectroscopy measurements based on kinetics, and molecular docking

Nanoparticles are intriguing researchers due to their unique properties. In this study, the Stöber method was used to obtain white particles. Through scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analysis, the spherical nano-scale particles were confirmed to be nano-silica. Bacteria adsorption experiment displayed that nano-silica could be accumulated on the surface of bacteria. The ultraviolet and fluorescence spectroscopy were used to explain the interaction between silica nanoparticles and recombinant protein A (S. aureus membrane protein) under simulated physiological conditions. The results of UV–vis and fluorescence experiments showed that the quenching mechanism between silica nanoparticles and recombinant protein A was static quenching. The key enzymes in bacterial cell wall synthesis (β-lactamase) and fatty acid biosynthesis (FabI and FabH) pathways were selected, and the molecular docking results explained that silica nanoparticles adhered to the surface of E. coli and S. aureus through forming hydrogen bond with amino acid residue.

Foliar water uptake of four shrub species in a semi-arid desert

Dew water plays an important role in supporting biological processes in desert ecosystems. Although some mechanisms underlying how plants survive in water-limited environments have been investigated, the water-use strategies of most desert plants remain unclear. We investigated whether foliar uptake of dew water occurs in four dominant shrub species (Artemisia ordosica, Hedysarum mongolicum, Salix psammophila, and Caragana korshinskii), in the Mu Us Desert of northern China. A fluorescent tracing experiment indicated that all four species absorbed water via leaf trichomes and cuticles, and A. ordosica also absorbed water via the stomata. A dew water exposure experiment and isotopic tracing revealed that water entered the leaves and stems of the four species, thereby enhancing their water potential. The relative contribution of dew water to the total water in the leaves of A. ordosica, H. mongolicum, S. psammophila, and C. korshinskii was 20.0 ± 7.0%, 8.4 ± 6.4%, 7.8 ± 1.9%, 3.1 ± 2.3%, respectively. The results demonstrated that all four species had the capacity to absorb water by their leaves, and the absorbed water could be transported to the stems. Our findings suggest that foliar water uptake may be one of the survival mechanisms by shrubs in the desert environment.

Towards ultimate bandwidth photon sources based on Compton backscattering: Design constraints due to nonlinear effects

Nuclear resonance fluorescence experiments typically require high rates of monochromatic photons due to the narrow linewidth of these resonances. Inverse Compton scattering sources are used to perform these experiments. Their intrinsic excellent monochromaticity is however spoiled by a variety of unavoidable imperfections related to the electron and laser beams. Some projects aim at reaching one per-mille of energy bandwidth, which requires attaining excellent brilliance of the electron beam but also a careful optimization of the laser-beam parameters. In particular, in such a situation, a careful accounting for the nonlinearities induced by a relatively large laser energy has to be considered. In this article, we revisit these nonlinearities with a quantum viewpoint with the goal to provide analytical expressions that can be employed for a very fast optimization of the performance of the source. These expressions were benchmarked against the CAIN event generator with an excellent accuracy in the parameters hypervolume that is of interest in this context. We also show that previously published expression often used to include laser nonlinearities in analytical bandwidth expressions significantly depart from the detailed CAIN simulations. The obtained expression are further used to optimize designs similar to those considered in on-going projects.

The Protective Benefit of Heme Oxygenase-1 Gene-Modified Human Placenta-Derived Mesenchymal Stem Cells in a N-Nitro-L-Arginine Methyl Ester-Induced Preeclampsia-Like Rat Model: Possible Implications for Placental Angiogenesis.

We previously reported that cytoprotective Heme oxygenase-1, HO-1 (HMOX1) gene-modified human placenta-derived mesenchymal stem cell (HO-1-PMSC) improved placental vascularization in vitro. In the current study, we explored the protective benefit of HO-1-PMSC transplantation in a preeclampsia (PE)-like rat model. A model of PE was successfully constructed by intraperitoneal injection of N-nitro-L-arginine methyl ester (L-NAME). Blood pressure and urinary protein levels were measured. Doppler ultrasound was examined to understand uteroplacental perfusion. ELISA was used to examine the serum levels of VEGF, PlGF, sFlt-1, and sEng. The placentas and fetuses were weighed to verify the improvement in pregnancy outcome. Immunohistochemical and H&E staining was used to detect microvessel density (MVD) in placental tissues and kidney pathology, respectively. The distribution of GFP-labeled PMSC in the placenta were observed under fluorescence microscopy. Blood pressure and proteinuria were reduced and kidney damage was improved. PE rat models treated with PMSC and HO-1-PMSC exhibited an increase in the quality of fetuses and placentas, MVD, VEGF, and PlGF expression, but substantially decreased expression of sFlt-1 and sEng. Doppler ultrasound showed that the placental perfusion was improved. Green fluorescent tracing experiments verified that the cells were successfully transplanted into the placenta and distributed in the blood vessels, indicating that the cells might participate in the process of angiogenesis. These results indicate that therapy with HO-1-PMSC could improve placental vascular dysplasia, increase placental perfusion, control PE symptoms, and promote pregnancy outcome by regulating the balance of angiogenic and antiangiogenic factors or directly participating in the repair of placental vessels in a PE-like rat model.

Thiocarbazone‐appended coumarin: An easily accessible ratiometric fluorescent chemosensor for multianalyte (Zn2+ and Cu2+) systems

AbstractA simple thiocarbazone‐appended coumarin derivative 1 with high selectivity as a ratiometric fluorescence sensor towards Cu2+ and Zn2+ in a semi‐aqueous solution was synthesised and characterised. Upon the addition of Cu2+ and Zn2+, the emission of 1 solution undergoes a blue and red shift to 480 and 545 nm according to the intense blue and yellow colour under a 365‐nm ultraviolet lamp, respectively. Binding was confirmed by ultraviolet‐visible absorption analysis, fluorescence measurement, mass spectrometry, proton nuclear magnetic resonance, Fourier Transform‐infrared spectroscopy and density functional theory calculation. The results show that 1 coordinated with Cu2+ and Zn2+ in different modes, resulting in different ratiometric fluorescence responses. The detection limits of the sensor 1 for Cu2+ and Zn2+ were 100.8 and 11.5 nM, respectively. The association constants (Ka) of Cu2+ and Zn2+ with 1 were determined to be 5.59 × 104 and 5.08 × 104 M−1, respectively. Fluorescent microscopy experiments demonstrated that probe 1 has the potential to be used to monitor Zn2+ and Cu2+ in living cells.

Endocytic Motif on a Biotin-Tagged HIV-1 Env Modulates the Co-Transfer of Env and Gag during Cell-to-Cell Transmission.

During HIV-1 transmission through T cell virological synapses, the recruitment of the envelope (Env) glycoprotein to the site of cell–cell contact is important for adhesion and for packaging onto nascent virus particles which assemble at the site. Live imaging studies in CD4 T cells have captured the rapid recruitment of the viral structural protein Gag to VSs. We explored the role of endocytic trafficking of Env initiated by a membrane proximal tyrosine motif during HIV transfer into target cells and examined the factors that allow Gag and Env to be transferred together across the synapse. To facilitate tracking of Env in live cells, we adapted an Env tagging method and introduced a biotin acceptor peptide (BAP) into the V4 loop of Env gp120, enabling sensitive fluorescent tracking of V4-biotinylated Env. The BAP-tagged and biotinylated HIVs were replication-competent in cell-free and cell-to-cell infection assays. Live cell fluorescent imaging experiments showed rapid internalized cell surface Env on infected cells. Cell–cell transfer experiments conducted with the Env endocytosis mutant (Y712A) showed increased transfer of Env. Paradoxically, this increase in Env transfer was associated with significantly reduced Gag transfer into target cells, when compared to viral transfer associated with WT Env. This Y712A Env mutant also exhibited an altered Gag/biotin Env fluorescence ratio during transfer that correlated with decreased productive cell-to-cell infection. These results may suggest that the internalization of Env into recycling pools plays an important role in the coordinated transfer of Gag and Env across the VS, which optimizes productive infection in target cells.