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Breast cancer (BC) is the most common malignancy among females worldwide, and its high metastasis rates are the leading cause of death just after lung cancer. Currently, tamoxifen (TAM) is a hydrophobic anticancer agent and a selective estrogen modulator (SERM), approved by the FDA that has shown potential anticancer activity against BC, but the non-targeted delivery has serious side effects that limit its ubiquitous utility. Therefore, releasing anti-cancer drugs precisely to the tumor site can improve efficacy and reduce the side effects on the body. Nanotechnology has emerged as one of the most important strategies to solve the issue of overdose TAM toxicity, owing to the ability of nano-enabled formulations to deliver desirable quantity of TAM to cancer cells over a longer period of time. In view of this, use of fluorescent carbon nanoparticles in targeted drug delivery holds novel promise for improving the efficacy, safety, and specificity of TAM therapy. Here, we synthesized biocompatible carbon nanoparticles (CNPs) using chitosan molecules without any toxic surface passivating agent. Synthesized CNPs exhibit good water dispersibility and emit intense blue fluorescence upon excitation (360 nm source). The surface of the CNPs has been functionalized with folate using click chemistry to improve the targeted drug uptake by the malignant cell. The pH difference between cancer and normal cells was successfully exploited to trigger TAM release at the target site. After six hours of incubation, CNPs released â¼ 74 % of the TAM drug in acidic pH. In vitro, studies have also demonstrated that after treatment with the synthesized CNPs, significant inhibition of the tumor growth could be achieved.
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Neoplasias de la Mama , Carbono , Portadores de Fármacos , Nanopartículas , Tamoxifeno , Tamoxifeno/farmacología , Tamoxifeno/química , Tamoxifeno/administración & dosificación , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Humanos , Femenino , Nanopartículas/química , Carbono/química , Portadores de Fármacos/química , Materiales Biocompatibles/química , Materiales Biocompatibles/farmacología , Animales , Línea Celular Tumoral , Sistemas de Liberación de Medicamentos , Células MCF-7 , Colorantes Fluorescentes/química , RatonesRESUMEN
White light emission (WLE), particularly from heteroatom free carbon dots (CDs), is unusual. Besides, deciphering the origin of WLE from a H-aggregated molecular fluorophore in such kinds of CDs is a challenging task due to their non-fluorescent character resulting from a forbidden transition from a lower-energy excitonic state. Therefore, rigorous investigation on their elusive excited state photophysical properties along with their steady-state optical phenomena has to be carried out to shed light on the nature of distinct emissive states formed in the CDs. Herein, for the first time, we report WLE from imperfect H-aggregates of co-facially π-π stacked humin-like structures comprising furfural monomer units as a unique molecular fluorophore in CDs, as revealed from combined spectroscopic and microscopic studies, synthesized through hydrothermal treatment of the single precursor, dextrose. H-aggregates in CDs show a broad range of excitation-dependent emission spectra with color coordinates close to pure white light, i.e., CIE (0.35, 0.37) and a color temperature of 6000 K. Imperfect orientation between the transition dipole moments of adjacent monomer units in the H-aggregate's molecular arrangement is expected to cause ground state symmetry breaking, as confirmed by Circular Dichroism (CD) studies, which established helically stacked nature in molecular aggregates and produced significant oscillatory strength at lower energy excitonic states to enable fluorescence. TRES and TAS investigations have been performed to minimise the intricacies associated with excited state photophysics, which is regarded as an essential step in gaining a grasp on emissive states. Based on the observation of two isoemissive spots in the time-resolved area normalized emission spectra (TRANES), the existence of three oligomeric species in the excited state equilibrium of the pure/hybrid H-aggregates has been established. The exciton dynamics through electron relaxation from the higher to the lower excitonic states, charge transfer (CT) states, and surface trap mediated emission in excimer states of H-aggregates have also been endorsed as three distinct emissive states from femtosecond transient absorption spectroscopy (TAS) studies corroborating with their steady-state absorption and emission behavior. The results would demonstrate the usage of CDs as a cutting-edge fluorescent material for creating aggregate-induced white light emission.
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Optical asymmetry and structural complexity across different length scales were realized in flower-shaped CuO nanostructures, prepared through refluxing an aqueous solution of copper acetate, sodium hydroxide, and D-tartaric acid, as well as in their toroid-like forms obtained on calcination at 600 °C. Atomic scale chirality in the flower morphology could be visualized as putative Boerdijk-Coexter-Bernal like tetrahelical fragments, while that in the toroid form could be identified as screw dislocation-driven helicity. The fraction of asymmetry in the nanostructures has been evaluated from their chiroptical responses based on Kuhn asymmetry factor (g) from circular dichroism (CD) spectroscopy in the entire UV-vis range. The origin of chirality in the two CuO nanostructures has been assigned to the helical arrangement of the Cu-O-Cu network in accordance with their microscopic and spectroscopic observations. Attempts have been made to interpret the crystallographic and geometric chiralities in the two CuO nanostructures based on the redshift and augmented intensity of the CD signal along with an increase in their corresponding anisotropic factor on calcination. Further, the diverse interaction of the toroid-shaped CuO nanostructures with enantiomeric tryptophan moieties has been illustrated from the measurement of their corresponding thermodynamic parameters.
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Fluorometric sensors for the detection of nerve agent mimics have received a lot of interest nowadays due to their high sensitivity and selectivity, ease of operation, and real-time monitoring. Pyridinic-N-rich carbon dots (NCDs) prepared through microwave-assisted pyrolysis of l-Malic acid and urea have been explored first time in this work as a novel turn-off fluorescent probe for the sensitive and selective detection of diethyl chlorophosphate (DCP), a nerve agent mimic. The as-prepared carbon dots contained a large amount of pyridinic nitrogen on their surface, which can modulate the photoluminescence properties of the NCDs. The blue emissive NCDs possessed both excitation wavelength-dependent and independent emission behavior. The detection of DCP was premised on quenching of the fluorescence emission intensity of NCDs in the presence of similar chemical reagents (e.g., trimethyl phosphate, triethyl phosphate, triethyl phosphonoacetate, triphenyl phosphate, diphenyl phosphate, tributyl phosphate). Fluorescence quenching of the NCDs in the presence of DCP has been attributed to the inner filter effect (IFE). From the linear Stern-Volmer plot (R2 = 0.9992), the limit of detection (LOD) was found to be 84 µM for sensing DCP for the concentration ranging between 3 and 15 mM. The biocompatibility of NCDs was assessed through cytotoxicity assay on MDA-MB-231 breast cancer cells. Fluorescence imaging demonstrated that NCDs have low cytotoxicity and can be employed successfully in cell imaging.
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Agentes Nerviosos , Puntos Cuánticos , Espectrometría de Fluorescencia/métodos , Carbono/química , Fluorometría , Puntos Cuánticos/química , Colorantes Fluorescentes/química , Nitrógeno/químicaRESUMEN
Defect engineering, such as modification of oxygen vacancy density, has been considered as an effective approach to tailor the catalytic performance on transition-metal oxide nanostructured surfaces. The role of oxygen vacancies (OV) on the surface of the as-prepared, zinnia-shaped morphology of CuO nanostructures and their marigold forms on calcination at 800 °C has been investigated through the study of model catalytic reactions of reduction of 4-nitrophenol and aerobic oxidation of benzyl alcohol. The OV on the surfaces of different morphologies of CuO have been identified and quantified through Rietveld analysis and HRTEM, EPR, and XPS studies. The structure-activity relationships between surface oxygen vacancies (OV) and catalytic performance have been systematically investigated. The enhanced catalytic performance of the cubic CuO nanostructures compared to their as-prepared forms has been attributed to the formation of surface oxygen species on the reactive and dominant (110) surface that has low oxygen vacancy formation energy. The mechanistic role of surface oxygen species in the studied reactions has been quantitatively correlated with the catalytic activity of the different morphological forms of the CuO nanostructures.
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INTRODUCTION In eukaryotic cells, the selective bidirectional transport of macromolecules between the nucleus and cytoplasm occurs through the nuclear pore complex (NPC). Embedded in nuclear envelope pores, the ~110-MDa human NPC is an ~1200-Å-wide and ~750-Å-tall assembly of ~1000 proteins, collectively termed nucleoporins. Because of the NPC's eightfold rotational symmetry along the nucleocytoplasmic axis, each of the ~34 different nucleoporins occurs in multiples of eight. Architecturally, the NPC's symmetric core is composed of an inner ring encircling the central transport channel and two outer rings anchored on both sides of the nuclear envelope. Because of its central role in the flow of genetic information from DNA to RNA to protein, the NPC is commonly targeted in viral infections and its nucleoporin constituents are associated with a plethora of diseases. RATIONALE Although the arrangement of most scaffold nucleoporins in the NPC's symmetric core was determined by quantitative docking of crystal structures into cryo-electron tomographic (cryo-ET) maps of intact NPCs, the topology and molecular details of their cohesion by multivalent linker nucleoporins have remained elusive. Recently, in situ cryo-ET reconstructions of NPCs from various species have indicated that the NPC's inner ring is capable of reversible constriction and dilation in response to variations in nuclear envelope membrane tension, thereby modulating the diameter of the central transport channel by ~200 Å. We combined biochemical reconstitution, high-resolution crystal and single-particle cryo-electron microscopy (cryo-EM) structure determination, docking into cryo-ET maps, and physiological validation to elucidate the molecular architecture of the linker-scaffold interaction network that not only is essential for the NPC's integrity but also confers the plasticity and robustness necessary to allow and withstand such large-scale conformational changes. RESULTS By biochemically mapping scaffold-binding regions of all fungal and human linker nucleoporins and determining crystal and single-particle cryo-EM structures of linker-scaffold complexes, we completed the characterization of the biochemically tractable linker-scaffold network and established its evolutionary conservation, despite considerable sequence divergence. We determined a series of crystal and single-particle cryo-EM structures of the intact Nup188 and Nup192 scaffold hubs bound to their Nic96, Nup145N, and Nup53 linker nucleoporin binding regions, revealing that both proteins form distinct question mark-shaped keystones of two evolutionarily conserved heterooctameric inner ring complexes. Linkers bind to scaffold surface pockets through short defined motifs, with flanking regions commonly forming additional disperse interactions that reinforce the binding. Using a structureguided functional analysis in Saccharomyces cerevisiae, we confirmed the robustness of linkerscaffold interactions and established the physiological relevance of our biochemical and structural findings. The near-atomic composite structures resulting from quantitative docking of experimental structures into human and S. cerevisiae cryo-ET maps of constricted and dilated NPCs structurally disambiguated the positioning of the Nup188 and Nup192 hubs in the intact fungal and human NPC and revealed the topology of the linker-scaffold network. The linker-scaffold gives rise to eight relatively rigid inner ring spokes that are flexibly interconnected to allow for the formation of lateral channels. Unexpectedly, we uncovered that linkerscaffold interactions play an opposing role in the outer rings by forming tight cross-link staples between the eight nuclear and cytoplasmic outer ring spokes, thereby limiting the dilatory movements to the inner ring. CONCLUSION We have substantially advanced the structural and biochemical characterization of the symmetric core of the S. cerevisiae and human NPCs and determined near-atomic composite structures. The composite structures uncover the molecular mechanism by which the evolutionarily conserved linkerscaffold establishes the NPC's integrity while simultaneously allowing for the observed plasticity of the central transport channel. The composite structures are roadmaps for the mechanistic dissection of NPC assembly and disassembly, the etiology of NPCassociated diseases, the role of NPC dilation in nucleocytoplasmic transport of soluble and integral membrane protein cargos, and the anchoring of asymmetric nucleoporins. [Figure: see text].
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Proteínas de Complejo Poro Nuclear , Poro Nuclear , Proteínas de Saccharomyces cerevisiae , Microscopía por Crioelectrón , Humanos , Modelos Moleculares , Poro Nuclear/química , Proteínas de Complejo Poro Nuclear/química , Conformación Proteica , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/químicaRESUMEN
Molybdenum disulfide (MoS2) nanosheets, due to having a highly active nature, being low cost and having unique physical and chemical properties, have shown their efficacy in the catalytic reduction of nitroarenes. Doping of transition metal ions in molybdenum disulfide (MoS2) nanosheets is a well-known strategy to enhance their catalytic efficiency for the reduction of nitroarenes, however, finding the optimum dopant amount is still a subject of ongoing research. Herein, we have synthesized few-layered cobalt (Co) doped MoS2 nanosheets with different cobalt content (2%, 4%, 6% and 8%) through the solvothermal approach, taking sodium molybdate dihydrate (Na2MoO4·2H2O), thiourea (CH4N2S) and cobalt acetate tetrahydrate [Co(CH3COO)2·4H2O] as precursors and their catalytic performance has been affirmed by monitoring the reduction of p-nitrophenol by NaBH4 in real time using UV-visible absorption spectroscopy. The 6% Co doped MoS2 nanosheets have exhibited superior catalytic activity with a pseudo-first order rate constant of 3.03 × 10-3 s-1 attributed to the abundant defects in the active edge sites having a dominant metallic 1T phase with Co ion activated defective basal planes, sulphur (S) edges, synergistic structural and electronic modulation between MoS2 and Co ions and enhanced electron transfer assisted through redox cycling in the active sites. An attempt has also been made to study the manipulation of structural and optical properties with cobalt doping in MoS2 nanosheets to establish a correlation between the catalytic efficiency and dopant content. This study demonstrates that proper tuning of Co doping in MoS2 nanosheets paves the way in searching for a potential alternative of a noble metal catalyst for the catalytic reduction of nitroarenes.
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Nitrogen and sulfur co-doped carbon dots (NSCDs) synthesized through one-pot microwave-assisted pyrolysis of tartaric acid and thioacetamide have been used as a fluorescent probe for the sensitive and selective detection of clinically important organic aldehyde cinnamaldehyde. The as-prepared NSCDs displayed blue fluorescence (â¼12% quantum yield), excellent aqueous solubility along with pH and excitation wavelength dependent emission behavior. In comparison to other aldehydes (formaldehyde, acetaldehyde, propionaldehyde, butyraldehyde, valeraldehyde, hexanal, crotonaldehyde and benzaldehyde) the fluorescence intensity of NSCDs was significantly quenched in the presence of cinnamaldehyde and the reduced intensity was linearly proportional to the concentration of cinnamaldehyde in the range of 0-15 mM with a detection limit of 99.0 µM. The fluorescence quenching of NSCDs was mainly attributed to the photo-excited electron transfer between NSCDs and aldehydes which was confirmed by measuring the life-time through time-resolved luminescence spectroscopy, energy levels of NSCDs through cyclic voltammetry (CV) and energy levels of aldehydes by density functional theory (DFT) based analyses. MTT assay of the NSCDs also proved their good biocompatibility and low toxicity towards human fibroblast cells thereby validating their suitability as a biologically relevant fluorescent probe for sensing cinnamaldehyde.
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Flagellated bacteria modulate their swimming behavior in response to environmental cues through the CheA/CheY signaling pathway. In addition to responding to external chemicals, bacteria also monitor internal conditions that reflect the availability of oxygen, light, and reducing equivalents, in a process termed "energy taxis." In Escherichia coli, the transmembrane receptor Aer is the primary energy sensor for motility. Genetic and physiological data suggest that Aer monitors the electron transport chain through the redox state of its FAD cofactor. However, direct biochemical data correlating FAD redox chemistry with CheA kinase activity have been lacking. Here, we test this hypothesis via functional reconstitution of Aer into nanodiscs. As purified, Aer contains fully oxidized FAD, which can be chemically reduced to the anionic semiquinone (ASQ). Oxidized Aer activates CheA, whereas ASQ Aer reversibly inhibits CheA. Under these conditions, Aer cannot be further reduced to the hydroquinone, in contrast to the proposed Aer signaling model. Pulse ESR spectroscopy of the ASQ corroborates a potential mechanism for signaling in that the resulting distance between the two flavin-binding PAS (Per-Arnt-Sim) domains implies that they tightly sandwich the signal-transducing HAMP domain in the kinase-off state. Aer appears to follow oligomerization patterns observed for related chemoreceptors, as higher loading of Aer dimers into nanodiscs increases kinase activity. These results provide a new methodological platform to study Aer function along with new mechanistic details into its signal transduction process.
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Proteínas Portadoras/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Flavina-Adenina Dinucleótido/metabolismo , Histidina Quinasa/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo/metabolismo , Proteínas Portadoras/química , Proteínas Portadoras/genética , Escherichia coli/química , Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Flavina-Adenina Dinucleótido/química , Flavina-Adenina Dinucleótido/genética , Histidina Quinasa/química , Histidina Quinasa/genética , Péptidos y Proteínas de Señalización Intercelular , Proteínas Quimiotácticas Aceptoras de Metilo/química , Proteínas Quimiotácticas Aceptoras de Metilo/genética , Oxidación-Reducción , Dominios ProteicosRESUMEN
Bacterial chemoreceptors associate with the histidine kinase CheA and coupling protein CheW to form extended membrane arrays that receive and transduce environmental signals. A receptor trimers-of-dimers resides at each vertex of the hexagonal protein lattice. CheA is fully activated and regulated when it is integrated into the receptor assembly. To mimic these states in solution, we have engineered chemoreceptor cytoplasmic kinase-control modules (KCMs) based on the Escherichia coli aspartate receptor Tar that are covalently fused and trimerized by a foldon domain (Tar(FO)). Small-angle X-ray scattering, multi-angle light scattering, and pulsed-dipolar electron spin resonance spectroscopy of spin-labeled proteins indicate that the Tar(FO) modules assemble into homogeneous trimers wherein the protein interaction regions closely associate at the end opposite to the foldon domains. The Tar(FO) variants greatly increase the saturation levels of phosphorylated CheA (CheA-P), indicating that the association with a trimer of receptor dimers changes the fraction of active kinase. However, the rate constants for CheA-P formation with the Tar variants are low compared to those for autophosphorylation by free CheA, and net phosphotransfer from CheA to CheY does not increase commensurately with CheA autophosphorylation. Thus, the Tar variants facilitate slow conversion to an active form of CheA that then undergoes stable autophosphorylation and is capable of subsequent phosphotransfer to CheY. Free CheA is largely incapable of phosphorylation but contains a small active fraction. Addition of Tar(FO) to CheA promotes a planar conformation of the regulatory domains consistent with array models for the assembly state of the ternary complex and different from that observed with a single inhibitory receptor. Introduction of Tar(FO) into E. coli cells activates endogenous CheA to produce increased clockwise flagellar rotation, with the effects increasing in the presence of the chemotaxis methylation system (CheB/CheR). Overall, the Tar(FO) modules demonstrate that trimerized signaling tips self-associate, bind CheA and CheW, and facilitate conversion of CheA to an active conformation.
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Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/química , Proteínas de la Membrana/química , Multimerización de Proteína , Receptores de Superficie Celular/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Histidina Quinasa , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Metiltransferasas/química , Metiltransferasas/genética , Metiltransferasas/metabolismo , Estructura Cuaternaria de Proteína , Estructura Terciaria de Proteína , Receptores de Superficie Celular/genética , Receptores de Superficie Celular/metabolismoRESUMEN
Dynamics are hypothesized to play an important role in the transmission of signals across membranes by receptors. Bacterial chemoreceptors are long helical proteins that consist of a periplasmic ligand-binding domain; a transmembrane region; a cytoplasmic HAMP (histidine kinase, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) domain; and a kinase-control module (KCM). The KCM is further composed of adaptation, hinge, and protein interaction regions (PIRs), the latter of which binds the histidine kinase CheA and adaptor CheW. Fusions of the Escherichia coli aspartate receptor KCM to HAMP domains of defined structure (H1-Tar vs. H1-2-Tar) give opposite responses in phosphotransfer and cellular assays, despite similar binding to CheA and CheW. Pulsed dipolar ESR spectroscopy (PDS) of these isolated on and off dimeric effectors reveals that, in the kinase-on state, the HAMP is more conformationally destabilized compared with the PIR, whereas in the kinase-off state, the HAMP is more compact, and the PIR samples a greater breadth of conformations. On and off HAMP states produce different conformational effects at the KCM junction, but these differences decrease through the adaptation region and into the hinge only to return with the inverted relationship in the PIR. Continuous wave-ESR of the spin-labeled proteins confirms that broader PDS distance distributions correlate with increased rates of dynamics. Conformational breadth in the adaptation region changes with charge alterations caused by modification enzymes. Activating modifications broaden the HAMP conformational ensemble but correspondingly, compact the PIR. Thus, chemoreceptors behave as coupled units, in which dynamics in regions proximal and distal to the membrane change coherently but with opposite sign.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Regulación Alostérica , Espectroscopía de Resonancia por Spin del Electrón , Escherichia coli , Proteínas de Escherichia coli , Histidina Quinasa , Proteínas Quimiotácticas Aceptoras de Metilo , Modelos Moleculares , Estabilidad Proteica , Estructura Secundaria de Proteína , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/química , Marcadores de SpinRESUMEN
HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between "on" and "off" conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.