Cancer-associated point mutations in isocitrate dehydrogenase 1 and 2 (IDH1, IDH2) confer a neomorphic enzymatic activity: the reduction of alpha-ketoglutarate (?KG) to D-2-hydroxyglutaric acid (2HG), which is proposed to act as an oncogenic metabolite by inducing hypermethylation of histones and DNA. While selective inhibitors of mutant IDH1 and IDH2 have been identified and are currently under investigation as potential cancer therapeutics, the mechanistic basis for their selectivity is not yet well-understood. A high-throughput screen for selective inhibitors of IDH1 bearing the oncogenic mutation R132H identified Compound 1, a bis-imidazole phenol that inhibits 2HG production in cells. We investigated the mode of inhibition of Compound 1 and a previously published IDH1 mutant inhibitor with a different chemical scaffold. Steady-state kinetics and biophysical studies show that both of these compounds selectively inhibit mutant IDH1 by binding to an allosteric site, and that inhibition is competitive with respect to Mg(2+). A crystal structure of Compound 1 complexed with R132H IDH1 indicates that the inhibitor binds at the dimer interface and makes a direct contact with a residue involved in binding of the catalytically essential divalent cation. These results show that targeting a divalent cation binding residue can enable selective inhibition of mutant IDH1, and suggest that differences in magnesium binding between wild-type and mutant enzymes may contribute to the inhibitors' selectivity for the mutant enzyme.
Intraductal administration of cytotoxic agents has been shown to inhibit the development of breast cancer in animal models. The object of this study was to demonstrate the safety of intraductal delivery cytotoxic agents in patients prior to mastectomy. This method is hopeful to be developed as a chemoprevention approach in patients with pre-malignant or non-invasive ductal lesions to prevent breast cancer which will be further developed.
Postchemotherapy relapse presents a major unmet medical need in acute myeloid leukemia (AML), where treatment options are limited. CD25 is a leukemic stem cell marker and a conspicuous prognostic marker for overall/relapse-free survival in AML. Rare occurrence of genetic alterations among PIM family members imposes a substantial hurdle in formulating a compelling patient stratification strategy for the clinical development of selective PIM inhibitors in cancer. Here we show that CD25, a bona fide STAT5 regulated gene, is a mechanistically relevant predictive biomarker for sensitivity to PIM kinase inhibitors. Alone or in combination with tyrosine kinase inhibitors, PIM inhibitors can suppress STAT5 activation and significantly shorten the half-life of MYC to achieve substantial growth inhibition of high CD25-expressing AML cells. Our results highlight the importance of STAT5 and MYC in rendering cancer cells sensitive to PIM inhibitors. Because the presence of a CD25-positive subpopulation in leukemic blasts correlates with poor overall or relapse-free survival, our data suggest that a combination of PIM inhibitors with chemotherapy and tyrosine kinase inhibitors could improve long-term therapeutic outcomes in CD25-positive AML.
A reversible detection method for vancomycin was developed utilizing the cantilever array sensor functionalized by a designed peptide consisting of a cysteine (Cys-), a space linker (-Gly-Gly-Gly-Gly-) and a molecular recognition ligand (-L-Lys-D-Ala-D-Ala). It was found that the peptide space linker was necessary and important for the response of the cantilever array sensor. The sensing cantilevers in the array were functionalized with the peptide while the reference cantilevers were modified by 6-mercapto-1-hexanol (MCH) to eliminate the influence of environmental disturbances. The binding between vancomycin and the peptide induced a change of surface stresses in the sensing cantilevers resulting in a differential deflection between the sensing and reference cantilevers. The reciprocal of the differential deflection is linear with the reciprocal of vancomycin concentration within the range of 2 ?M to 100 ?M (R=0.993) at a detection limit of 0.2 ?M (S/N=3). The reversible detection can be realized just by regenerating the sensing cantilevers with running buffer solution. Other antibiotics such as doxycycline, streptomycin, and kanamycin have negligible effect on the response of the sensor. The sensor can also be utilized for reversible detection of vancomycin in serum background, which clearly indicates the potential of the sensor for vancomycin detection in real biological samples.
A biosensor has been developed with a photonic crystal structure used in a total-internal-reflection (PC-TIR) configuration for label-free detection of a cardiac biomarker: Troponin I (cTnI). In contrast to a conventional optical microcavity that has a closed structure with its cavity layer sandwiched between two high-reflection surfaces, the PC-TIR configuration creates a unique open microcavity, which allows its cavity layer (sensing layer) to be easily functionalized and directly exposed to analyte molecules for bioassays. In this study, a PC-TIR sensor has been used for the label-free measurements of cardiac biomarkers by monitoring the changes in the resonant condition of the cavity due to biomolecular binding processes. Antibodies against cTnI are immobilized on the sensor surface for specific detection of cTnI with a wide range of concentrations. Detection limit of cTnI with a concentration as low as 0.1ngmL(-1) has been achieved.
Human gonadotropin-releasing hormone receptor (GnRH-R; or type I GnRH-R), which is expressed in tumor cells, has gained more and more attention as a specific target for cancer therapy. Given the clinical utility, the improved characterization of both the subcellular distribution and surface organization of GnRH-R is an important step in the development of more effective and possibly new therapeutic strategies. In the present study, the nano-organization of human GnRH-R was analyzed on fixed human bladder cancer cells (T24) by atomic force microscopy (AFM). The recognition images reveal that GnRH-Rs have a tendency to assemble in nanodomains (or clusters) that are irregularly distributed on the T24 cell surface. The locations of the GnRH-Rs were identified on the topographical images with nanometer accuracy. The obtained results enrich our understanding of the local distribution of GnRH-Rs on the bladder cancer cell membrane and demonstrate the ability of biological AFM to provide more complete and exact information at the single molecule level.
A facile simple hydrothermal method combined with a post-solution reaction is developed to grow interconnected three dimensional (3D) hierarchical Co-Al layered double hydroxides (LDHs) on reduced graphene oxide (rGO). The obtained 3D hierarchical rGO-LDHs are characterized by field emission scanning electron microscopy, X-ray diffraction, and X-ray photo-electron spectroscopy. As LDHs nanosheets directly grow on the surface of rGO via chemical covalent bonding, the rGO could provide facile electron transport paths in the electrode for the fast Faradaic reaction. Moreover, benefiting from the rational 3D hierarchical structural, the rGO-LDHs demonstrate excellent electrochemical properties with a combination of high charge storage capacitance, fast rate capability and stable cycling performance. Remarkably, the 3D hierarchical rGO-LDHs exhibit specific capacitance values of 599 F g(-1) at a constant current density of 4 A g(-1). The rGO-LDHs also show high charge-discharge reversibility with an efficiency of 92.4% after 5000 cycles.
The small bowel is one of the critical organs involved in gastrointestinal complications in cervical cancer treated with postoperative intensity modulated radiotherapy. Even with modest doses of radiation therapy (45-50Gy), the risk of severe injury from postoperative radiation therapy is between 5% and 15%. Up to now, a predictive model of acute GI complications of the small bowel has been established with the aid of Quantitative Analyses of Normal Tissue Effects in the Clinic. However, the correlation between dose-volume effect and chronic GI complications of the small bowel has not been extensively investigated. In the article, the correlation has been studied preliminarily.
A label-free detection method of kanamycin using aptamer-based cantilever array sensor was developed. The cantilever array was composed of sensing cantilevers and reference cantilevers. This configuration allowed direct detection of individual cantilever deflections and subsequent determination of differential deflection of sensing/reference cantilever pair. The sensing cantilevers were functionalized with kanamycin aptamer, which was used as receptor molecules while the reference cantilevers were modified with 6-mercapto-1-hexanol (MCH) to eliminate the influence of environmental disturbances. The kanamycin-aptamer interaction induced a change in cantilever surface stress, which caused a differential deflection between the sensing and reference cantilever pair. The surface stress change was linear with kanamycin concentration over the range of 100 ?M-10mM with a correlation coefficient of 0.995. A detection limit of 50 ?M was obtained, at a signal-to-noise ratio of 3. The sensor also showed good selectivity against other antibiotics such as neomycin, ribostamycin and chloramphenicol. The facile method for kanamycin detection may have great potential for investigating more other molecules.
MicroRNA (miRNA) 200s regulate E-cadherin by directly targeting ZEB1/ZEB2, which are transcriptional repressors of E-cadherin. Decreased expression of E-cadherin results in cancer cells losing interaction with the extracellular matrix and detaching from the primary tumor. Normally, cells will undergo anoikis after losing interaction with the extracellular matrix. Cancer cells must, therefore, possess the ability to resist anoikis during the process of metastasis. Here we show that miRNA-200b regulates anoikis by directly targeting the 3 UTR of Pin1 mRNA and regulating Pin1 expression at the translational level. We found that down-regulation of miRNA-200b promotes cancer cells survival during metastasis, and the homeless state of these cells resulted in decreased expression of miRNA-200b in the MCF-7 cell line. We also found that expression of miRNA-200b is down-regulated in human breast cancer during lymph node metastasis, which has a significant negative correlation with Pin1 expression. Two members of the ETS (E-26) family (PEA3 and ELK-1) regulate the expression of miRNA-200b. PEA3 promotes the expression of miRNA-200b, and ELK-1 is a transcriptional repressor of miRNA-200b. In addition, miRNA-200b regulates the activity of PEA3 and ELK-1 via the Pin1-pERK pathway and forms self-regulated feedback loops. This study characterizes the role of miRNA-200b in the regulation of anoikis and demonstrates the regulation of its own expression in the process of metastasis.
PRP4 kinase is known for its roles in regulating pre-mRNA splicing and beyond. Therefore, a wider spectrum of PRP4 kinase substrates could be expected. The role of PRP4 kinase in cancer is also yet to be fully elucidated. Attaining specific and potent PRP4 inhibitors would greatly facilitate the study of PRP4 biological function and its validation as a credible cancer target. In this report, we verified the requirement of enzymatic activity of PRP4 in regulating cancer cell growth and identified an array of potential novel substrates through orthogonal proteomics approaches. The ensuing effort in structural biology unveiled for the first time unique features of PRP4 kinase domain and its potential mode of interaction with a low molecular weight inhibitor. These results provide new and important information for further exploration of PRP4 kinase function in cancer.
We present a new method for sensitive ultrasound detection using an open-cavity optoacoustic sensor. Our results have demonstrated significant enhancement of detection sensitivity when the open-cavity sensor is used in media with large isothermal compressibility. A near-linear relationship between detected optoacoustic signal strength and isothermal compressibility has been found.
The understanding of ligand binding interactions is an important component of understanding the fundamental mechanism of receptor function. In this study, the binding abilities of EGF and TGF-? to EGFR on human bladder cancer (T24) cells were investigated by single molecular force spectroscopy (SMFS) based on atomic force microscopy (AFM). By approaching the specifically functionalized AFM tips to the T24 cell surface and subsequent retraction, specific unbinding events of the EGF/EGFR complexes and TGF-?/EGFR complexes were investigated. Further, the unbinding forces and the kinetic off rate constants that govern the bond stabilities were calculated through varying the external mechanic forces applied. Meanwhile, the distances from the energy minimum to the transition states along the separation paths of the EGF/EGFR complexes and TGF-?/EGFR complexes were deduced. This study at single-molecule level may enrich our understanding of the ligand binding properties of EGFR and provide some new information to the development of improved EGFR inhibitors. In addition, the results present new insight into the study of the energy landscape of the dissociation of ligand-EGFR system.
A facile two step process was developed for the synthesis of porous Co3O4 nanorods-reduced graphene oxide (PCNG) hybrid materials based on the hydrothermal treatment cobalt acetate tetrahydrate and graphene oxide in a glycerol-water mixed solvent, followed by annealing the intermediate of reduced graphene oxide-supported Co(CO3)0.5(OH)·0.11H2O nanorods in a N2 atmosphere. The morphology and microstructure of the composites were examined by X-ray diffraction, X-ray photoelectron spectroscopy, transmission electron microscopy and Raman spectroscopy. It is shown that the obtained PCNG have intrinsic peroxidase-like activity. The PCNG are utilized for the catalytic degradation of methylene blue. The good catalytic performance of the composites could be attributed to the synergy between the functions of porous Co3O4 nanorods and reduced graphene oxide.
A facile method is proposed for the synthesis of three-dimensional (3D) flower-like Co3-xFexO4 ferrite (CF) hollow spheres, using SiO2@FeOOH as precursor. The CF hollow spheres are efficient for the catalytic degradation of methylene blue (MB) in the presence of H2O2 at 80 °C. The obtained CF hollow spheres were characterized using transmission electron microscopy, field emission scanning electron microscopy, X-ray diffraction, X-ray photo-electron spectroscopy, and N2 adsorption-desorption isotherm measurements. The formation of 3D hierarchical flower-like superstructure was influenced by the relative amount of urea used. As the mole ratio of CoCl2 and urea decreased, the structure of the products was tailored from yolk-like spheres to hollow spheres with different sized void interiors. Moreover, N2 adsorption-desorption isotherm analysis showed that the CF hollow spheres have a large specific surface area (163 m(2) g(-1)) which provided more activity sites. The CF hollow spheres can catalyze the oxidation of MB efficiently. These results indicate that the designed CF hollow spheres exhibit promising capability for the degradation of dyes.
Glucokinase (GK) acts as a glucose sensor by facilitating glucose phosphorylation into glucose-6-phosphate (G6P) in the liver and pancreas, the two key target tissues. LCZ960, a glucokinase activator exerts a stimulatory effect on GK activity in hepatocytes in vitro. This study aimed to verify in vivo that LCZ960 stimulates glucose uptake primarily through a mechanism involving hepatic GK activation. Acute and chronic LCZ960 treatment-induced changes in glycemia and hepatic glucose turnover were measured in high fat diet-induced obese (DIO) mice and rats. G6P production and glycogen cycling were quantified by (13)C-MR spectroscopy during a [1-(13)C]glucose infusion, followed by a pulse-chase with [(12)C]glucose to mimic postprandial conditions in rats. Acute treatment with LCZ960 dose-dependently reduced blood glucose without causing hypoglycemia in DIO mice. Chronic LCZ960 treatment maintained normoglycemia and improved glucose tolerance without increased insulin secretion in DIO mice and rats. In rats, LCZ960 stimulated a 240% increase (P<0.05) in the glycogen synthase flux. However, due to a much higher glycogen breakdown (LCZ960: 48 ± 15 vs control: 4 ± 1?mol/kg/min, P<0.05), this translated to only a 46% (ns) increase in glycogen storage (Vsyn net, LCZ960: 64±26 vs control: 43 ± 6 ?mol/kg/min). Despite a 4-fold increase in hepatic glycogen turnover (LCZ960: 36.0 ± 5.5% vs control: 8.3 ± 2.0%), LCZ960 did not impact glucose-stimulated G6P accumulation. LCZ960 did not cause hypoglycemia in DIO rodents. Under hyperglycemic conditions, LCZ960 induced a robust increase in hepatic glycogen cycling. Since net hepatic glycogen storage is diminished in type 2 diabetes patients, stimulation of glycogen synthesis may contribute to the anti-hyperglycemic properties of glucokinase activation.
Toll-like receptor (TLR) signaling is a key component of innate immunity. Aberrant TLR activation leads to immune disorders via dysregulation of cytokine production, such as IL-12/IL-23. Herein, we identify and characterize PIKfyve, a lipid kinase, as a critical player in TLR signaling using apilimod as an affinity tool. Apilimod is a potent small molecular inhibitor of IL-12/IL-23 with an unknown target and has been evaluated in clinical trials for patients with Crohns disease or rheumatoid arthritis. Using a chemical genetic approach, we show that it binds to PIKfyve and blocks its phosphotransferase activity, leading to selective inhibition of IL-12/IL-23p40. Pharmacological or genetic inactivation of PIKfyve is necessary and sufficient for suppression of IL-12/IL-23p40 expression. Thus, we have uncovered a phosphoinositide-mediated regulatory mechanism that controls TLR signaling.
We present a new method for specific detection of oxytetracycline (OTC) at nanomolar concentrations based on a microfabricated cantilever array. The sensing cantilevers in the array are functionalized with self-assembled monolayers (SAMs) of OTC-specific aptamer, which acts as a recognition molecule for OTC. While the reference cantilevers in the array are functionalized with 6-mercapto-1-hexanol SAMs to eliminate the influence of environmental disturbances. The cantilever sensor shows a good linear relationship between the deflection amplitude and the OTC concentration in the range of 1.0-100 nM. The detection limit of the cantilever array sensor is as low as 0.2 nM, which is comparable to some traditional methods. Other antibiotics such as doxycycline and tetracycline do not cause significant deflection of the cantilevers. It is demonstrated that the cantilever array sensors can be used as a powerful tool to detect drugs with high sensitivity and selectivity.
We report a simple and sensitive method for label-free detection of single-stranded DNA-binding protein (SSBP) based on an array of microfabricated cantilevers. The single-stranded DNA (ssDNA) was immobilized on the surface of the sensing cantilevers to detect SSBP, while the reference cantilevers were modified with 6-mercapto-1-hexanol to detect any unwanted cantilever deflection. The differential deflection signals that reveal specific SSBP-ssDNA binding have been found to depend on the SSBP concentration. Using the cantilever array sensor we can detect SSBP in the concentration range from 0.01 to 7 ?g mL(-1). Other proteins, such as thrombin or bovine serum albumin induced no significant deflection of the cantilevers. Our results show the potential for the application of cantilever array sensor system as a powerful tool to detect proteins with high sensitivity and specificity.
Molecular recognition force spectroscopy (MR-FS) was applied to investigate the dynamic interaction between aptamer GBI-10 and tenascin-C (TN-C) on human glioblastoma cell surface at single-molecule level. The unbinding force between aptamer GBI-10 and TN-C was 39?pN at the loading rate of 0.3?nN?sec?¹. A series of kinetic parameters concerning interaction process such as the unbinding force f(u) , the association rate constant k(on) , dissociation rate constant at zero force k(off) , and dissociation constant K(D) for aptamer GBI-10/TN-C complexes were acquired. In addition, the interaction of aptamer GBI-10 with TN-C depended on the presence of Mg²?. This work demonstrates that MR-FS can be used as an attractive tool for exploring the interaction forces and dynamic process of aptamer and ligand at the single-molecule level. As a future perspective, MR-FS may be used as a potential diagnostic and therapeutic tool by combining with other techniques.
Bacillus anthracis has posed a threat of becoming biological weapons of mass destruction due to its virulence factors encoded by the plasmid-borne genes, such as lef for lethal factor. We report the development of a fast and sensitive anthrax DNA biosensor based on a photonic crystal structure used in a total-internal-reflection configuration. For the detection of the lef gene, a single-stranded DNA lef probe was biotinylated and immobilized onto the sensor via biotin-streptavidin interactions. A positive control, lef-com, was the complementary strand of the probe, while a negative control was an unrelated single-stranded DNA fragment from the 16S rRNA gene of Acinetobacter baumannii. After addition of the biotinylated lef probe onto the sensor, significant changes in the resonance wavelength of the sensor were observed, resulting from binding of the probe to streptavidin on the sensor. The addition of lef-com led to another significant increase as a result of hybridization between the two DNA strands. The detection sensitivity for the target DNA reached as low as 0.1 nM. In contrast, adding the unrelated DNAs did not cause an obvious shift in the resonant wavelength. These results demonstrate that detection of the anthrax lef by the photonic crystal structure in a total-internal-reflection sensor is highly specific and sensitive.
A novel third-generation hydrogen peroxide (H(2)O(2)) biosensor was developed by immobilizing horseradish peroxidase (HRP) on a biocompatible attapulgite (ATP) modified glassy carbon (GC) electrode. The ATP could provide a biocompatible microenvironment for enzyme molecules, greatly amplify the coverage of HRP molecules on the electrode surface, and most importantly facilitate the direct electron transfer between HRP and the electrode. The biosensor construction process was followed by atomic force microscopy (AFM). Cyclic voltammetry was employed to characterize the properties of the biosensor. A linear calibration plot of the enzyme electrode was obtained over the range of 5 µM to 0.3 mM for H(2)O(2) with a detection limit of 5 µM. Furthermore, the biosensor showed high sensitivity, good reproducibility, and fine long-term stability.
A novel strategy to fabricate hydrogen peroxide (H(2)O(2)) sensor was developed by electrodepositing Ag nanoparticles (NPs) on a glassy carbon electrode modified with natural nano-structure attapulgite (ATP). The result of electrochemical experiments showed that such constructed sensor had a favorable catalytic ability to reduce H(2)O(2). The good catalytic activity of the sensor was ascribed to the ATP that facilitated the formation and homogenous distribution of small Ag NPs. The resulted sensor achieved 95% of the steady-state current within 2s and had a 2.4 ?M detection limit of H(2)O(2).
Ag-graphene composite nanosheets (AGCN) with adjustable size and well-controlled densities of Ag nanoparticles (Ag NPs) using Poly(N-vinyl-2-pyrrolidone) (PVP) as a reductant and stabilizer are reported. The obtained AGCN substrate is extremely suitable for surface-enhanced Raman spectroscopy (SERS).
Carbohydrates are involved in many essential biological recognition processes in physiological and pathological states. Thus, it is important to understand the mechanism of protein-carbohydrate interactions at molecular level. In the present study, molecular recognition force spectroscopy was applied to investigate the interactions between RCA???, a lectin from Ricinus communis, and galactose (Gal) and asialofetuin (ASF) at the single-molecule level. RCA??? coupled to the AFM tip could specifically recognize Gal and ASF, respectively. The unbinding forces of RCA???-Gal and RCA???-ASF increase linearly with the logarithm of loading rate. The results reveal that the binding capability of RCA??? toward Gal is weaker than that of ASF, implicating a multivalent effect in the RCA???-ASF interaction.
An electrochemical microcantilever (EMC) was used to study the intermolecular interaction of self-assembly monolayers (SAMs) with different n-alkanethiols chain lengths (n = 0, 4, 6, 8, 12, 16) on a Au-coated microcantilever surface. Comparing potential cycling and steps in NaClO(4) solution within the same potential range, the deflection rate of bare microcantilevers is much smaller for the former which revealed that potential excitation, i.e. the surface charge, played the dominant role in driving the instant and large deflection of the bare microcantilever, while the smaller deflection amplitude of the former implied that adsorption of ClO(4)( - ) had an adverse effect on the potential-induced stress. Upon adsorption of SAMs, the deflection amplitude of the microcantilever under the potential step was much smaller than that of a bare microcantilever, and linearly decreased with the chain length increasing for n ? 8 (the linear correlation coefficient and the slope are 0.98 and about - 10.4 nm per CH(2) unit, respectively), following a transition (8 ? n ? 12) to a stable state (n ? 12). The decrease of deflection amplitude and faster decay of deflection rate of the SAMs modified microcantilever under the potential step implyed increasing compactness of the SAMs with longer chains.
Hydroxyapatite (HA) coatings on titanium (Ti) substrates have attracted much attention owing to the combination of good mechanical properties of Ti and superior biocompatibility of HA. Incorporating silver (Ag) into HA coatings is an effective method to impart the coatings with antibacterial properties. However, the uniform distribution of Ag is still a challenge and Ag particles in the coatings are easy to agglomerate, which in turn affects the applications of the coatings. In this study, we employed pulsed electrochemical deposition to co-deposit HA and Ag simultaneously, which realized the uniform distribution of Ag particles in the coatings. This method was based on the use of a well-designed electrolyte containing Ag ions, calcium ions and l-cysteine, in which cysteine acted as the coordination agent to stabilize Ag ions. The antibacterial and cell culture tests were used to evaluate the antibacterial properties and biocompatibility of HA/Ag composite coatings, respectively. The results indicated the as-prepared coatings had good antibacterial properties and biocompatibility. However, an appropriate silver content should be chosen to balance the biocompatibility and antibacterial properties. Heat treatments promoted the adhesive strength and enhanced the biocompatibility without sacrificing the antibacterial properties of the HA/Ag coatings. In summary, this study provided an alternative method to prepare bioactive surfaces with bactericidal ability for biomedical devices.
To improve the practicability of rapid biochemical oxygen demand (BOD) method, we proposed a stable BOD sensor based on immobilizing multi-species BODseed for wastewater monitoring in the flow system. The activation time of the biofilm was greatly shortened for the biofilm prepared by BODseed in the organic-inorganic hybrid material. Some influence factors such as temperature, pH, and concentration of phosphate buffer solution (PBS) were investigated in detail in which high tolerance to environment was validated for the BOD sensor permitted a wide pH and PBS concentration ranges. The minimum detectable BOD was around 0.5 mg/l BOD under the optimized 1.0 mg/ml BODseed immobilized concentration. The as-prepared BOD sensor exhibited excellent stability and reproducibility for different samples. Furthermore, the as-prepared BOD biosensor displayed a notable advantage in indiscriminate biodegradation to different organic compounds and their mixture, similar to the character of conventional BOD(5) results. The results of the BOD sensor method are well agreed with those obtained from conventional BOD(5) method for wastewater samples. The proposed rapid BOD sensor method should be promising in practical application of wastewater monitoring.
Dynamics of fluorescent diamond nanoparticles in HeLa cells has been studied with two-photon fluorescence correlation spectroscopy (FCS). Fluorescent nanodiamond (FND) is an excellent fluorescent probe for bioimaging application, but they are often trapped in endosomes after cellular uptake. The entrapment prohibits FCS from being performed in a time frame of 60 s. Herein, we show that the encapsulation of FNDs within a lipid layer enhances the diffusion of the particles in the cytoplasm by more than one order of magnitude, and particles as small as 40 nm can be probed individually with high image contrast by two-photon excited luminescence. The development of the technique together with single particle tracking through one-photon excitation allows probing of both short-term and long-term dynamics of single FNDs in living cells.
We systematically validated a robust 96-well Caco-2 assay via an extended set of 93 marketed drugs with diverse transport mechanisms and quantified by LC/MS/MS, to investigate its predictive utility while dealing with challenging discovery compounds. Utilizing nonlinear fit, the validation led to a good correlation (R(2) = 0.76) between absorptive permeability, log P(app)(A-B), from in vitro Caco-2 assay and reported human fraction of dose absorbed. We observed that paracellular compounds could be flagged by log P(app)(A-B) (<-5.5 cm/s) and physicochemical property space (c log P < 1). Of 8000 Novartis discovery compounds examined 13% were subject to low recovery (<30%). Compound loss was investigated by comparing cell monolayer and artificial membrane, while 0.5% bovine serum albumin (in both donor and acceptor compartments) was utilized to improve recovery. The second focus of this study was to investigate the advantages and limitations of the current Caco-2 assay for predicting in vivo intestinal absorption. Caco-2 measurements for compounds with high aqueous solubility and low in vitro metabolic clearance were compared to 88 in vivo rat bioavailability studies. Despite the challenges posed by discovery compounds with suboptimal physicochemical properties, Caco-2 data successfully projected low intestinal absorption. This platform sets the stage for mechanistically evaluating compounds towards improving in vitro-in vivo correlations.
Monitoring biochemical oxygen demand (BOD) by mediator method (BOD(Med)) has been developed for recent years and deaerated condition was generally adopted to avoid the effect of oxygen, but the deaerated condition was unfavorable in practical applications. Herein, we first proposed another way to explore non-deaerated BOD(Med) (called NDA-BOD(Med)) method utilizing ferricyanide, which was reduced by Escherichia coli upon catalyzing organic substrate to produce ferrocyanide. We attempted to explain the feasibility of NDA-BOD(Med) by the two aspects. Firstly, the obtained biodegradation efficiencies of the bacteria under the deaerated and non-deaerated conditions were similar, and the concentration of O(2) (0.25mM at 8mg/L O(2)) is 1-2 order of magnitude lower than that of mediator commonly used (55mM ferricyanide), so the effect of O(2) to measurements could be neglected. Secondly, the relationship between the artificial and the natural electron acceptor was investigated, and it was found that the oxygen consumption in the NDA-BOD(Med) measurement was mainly contributed to endogenous values. Furthermore, the performance of present NDA-BOD(Med) was reported, and this method was optimized for measuring the low-concentration samples, synthetic wastewater and real polluted wastewater. The NDA-BOD(Med) provides a simple and efficient way in rapid BOD determinations, especially advantageous for in situ monitoring of water system.
(2 + 1) Resonance enhanced multiphoton ionization (REMPI) spectra were recorded for 2-butanone to study its photoionization dynamics. Two-photon excitation (53,200-55,000 cm(-1) and 57,000-59,500 cm(-1)) was used to prepare the molecule in the 3s and 3p Rydberg states, respectively. Vibrational transitions in the spectrum were assigned with the aid of ab initio calculations as well as photoelectron imaging results. Photoelectron imaging data in the 3s Rydberg region exhibits a vibrational progression in the CCOC deformation mode in the ionic state superimposed on an otherwise diagonal (delta v = 0) ionization. Photoelectron imaging data in the 3p Rydberg region shows 3p-3s Rydberg-Rydberg mixing. The ionization energy obtained directly from the 3p photoelectron imaging is 9.541 eV.
Fluorescent nanodiamonds (FNDs) are nontoxic and photostable nanomaterials, ideal for long-term in vivo imaging applications. This paper reports that FNDs with a size of approximately 140 nm can be covalently conjugated with folic acid (FA) for receptor-mediated targeting of cancer cells at the single-particle level. The conjugation is made by using biocompatible polymers, such as polyethylene glycol, as crosslinked buffer layers. Ensemble-averaged measurements with flow cytometry indicate that more than 50% of the FA-conjugated FND particles can be internalized by the cells (such as HeLa cells) through receptor-mediated endocytosis, as confirmed by competitive inhibition assays. Confocal fluorescence microscopy reveals that these FND particles accumulate in the perinuclear region. The absolute number of FNDs internalized by HeLa cells after 3 h of incubation at a particle concentration of 10 microg mL(-1) is in the range of 100 particles per cell. The receptor-mediated uptake process is further elucidated by single-particle tracking of 35-nm FNDs in three dimensions and real time during the endocytosis.
A new approach to one-dimensional organization of gold nanoparticles (2-4 nm) is described, using poly(4-vinylpyridine) (P4VP) molecular chain as a template with the mediation of free Cu2+ ion coordination. The assembly was conducted on freshly prepared mica surfaces and in aqueous solution, respectively. The surface assembly was characterized by tapping mode atomic force microscopy (AFM), observing the physisorbed molecules in their chain-like conformation with an average height of 0.4 nm. By the mediation of Cu2+ ions, gold nanoparticles modified by 3-mercaptopropionic acid were deposited onto the molecular chains, evidenced by a clear increase in height. Generation of the network in solution is time-dependent and pH reversible, characterized by UV-vis absorption spectra and transmission electron microscopy (TEM). No comparable network is obtained without Cu2+ ions, indicating the significance of ionic mediation. A mechanism for the self-assembly in solution is proposed, and the nature of the mediation of Cu2+ ions was identified by x-ray photoelectron spectroscopy (XPS).
The replacement of coronene monolayer on Au (111) by 6-mercapto-1-hexanol (MHO) was studied by in situ scanning tunneling microscopy (STM) in solutions. It was found that the rate of replacement depends strongly on the concentration of MHO. The replacement finished within a second at a higher concentration of MHO. At a lower concentration, the slow replacement could be followed by in situ STM. The replacement occurred initially near the elbow position of reconstructed Au (111) with the formation of pits in a single or several missing molecules. With the proceeding of replacement, these small pits expanded, and the surrounding coronene molecules were gradually substituted by MHO, which developed into ordered domains within a spatial confined environment. Meanwhile, the reconstruction of Au (111) was lifted. The replacement expanded fast along the reconstruction lines in the domain. For the fast replacement, a ( radical3 x radical3) R30 degrees adlattice was observed, while a c(4 x 2) superlattice was observed for the slow replacement. The close-packed phase of MHO self-assembled monolayers (SAMs) appeared directly instead of a flat-lying phase, which implied that the coronene molecules resist the direct contact of hydrocarbon chain of MHO with Au (111) surface. The replacing rate of the overall process exhibited a signoidal shape with time.
In the present study, platinum nanoparticles modified with Prussian blue (PB) have been synthesized by a heterogeneous catalytic reaction. Transmission electronic microscopy (TEM) confirmed the deposition of nanoclusters around the surfaces of platinum particles, and spectroscopic studies verified that the molecular composition of the nanoclusters was dominantly PB and a minority of platinum ferricyanide. Thus, it was shown that the platinum particles behaved not only as catalysts for the growth of PB, but also as a reactant to generate a PB analogue complex. Moreover, potassium was not detected in the final product, demonstrating that the molecular nature of PB was in the "water-insoluble" form, Fe(4)(3+)[Fe(II)(CN)(6)](3).
A hybrid material based on Pt nanoparticles (Pt NPs) and multi-walled carbon nanotubes (MWNTs) was fabricated with the assistance of PEI and formic acid. The cationic polyelectrolyte PEI not only favored the homogenous dispersion of carbon nanotubes (CNTs) in water, but also provided sites for the adsorption of anionic ions PtCl(4)(2-) on the MWNTs sidewalls. Deposition of Pt NPs on the MWNTs sidewalls was realized by in situ chemical reduction of anionic ions PtCl(4)(2-) with formic acid. The hybrid material was characterized with TEM, XRD and XPS. Its excellent electrocatalytic activity towards both oxygen reduction in acid media and dopamine redox was also discussed.
We present a detailed photoion and photoelectron imaging study of isobutanal cation dynamics. The 2 + 1 REMPI spectrum via the (n,3s) Rydberg transition was recorded and analyzed under both cold and warm beam conditions in an effort to identify the predicted trans conformer. Photoelectron imaging was used to establish the ion energetics accurately and to aid in the assignment of the REMPI spectra. On the basis of the photoelectron spectra and ab initio calculations, a peak at 54336.8 cm(-1) is assigned to the trans conformer. We have also assigned the photoelectron spectra and identified some hot band transitions not reported in previous work. We determined the adiabatic ionization energy to be 9.738 eV. We also studied the photodissociation dynamics of isobutanal cations leading to several product channels, but no evidence of vibrational mode or conformational isomer dependence on either the product branching or dynamics was seen for this system.
The adsorption of dopamine (DA) molecules on gold and their interactions with Fe3+ were studied by a microcantilever in a flow cell. The microcantilever bent toward the Au side with the adsorption of DA due to the change of surface stress induced by the intermolecular hydrogen bonds of DA or the charge transfer effect between adsorbates and the substrate. The interaction process between DA adsorbates and Fe3+ was revealed by the deflection curves of microcantilever. As indicated by the appearance of a variation during the decline of curves, two steps were observed in the curve at relative high concentrations of Fe3+. In this case, Fe3+ reacted with DA molecules only in the outer layers and the complexes removed with solution. Then Fe3+ reacted further with DA molecules forming the surface complex in the first layer next to the gold. At this stage, the stability of surface complexes was time dependent, i.e., unstable initially and stable finally. This may be due to the surface complexes change from mono-dentate to bi-dentate complexes. In another case, i.e., at relative low concentration of Fe3+, only the first step was observed as indicated by the absence of a variation. X-ray photoelectron spectroscopy (XPS) and cycling voltammetry (CV) results provided complementary evidence for the result of microcantilever and proposal. As low as 5 x 10(-10) M Fe3+ was detected by DA modified microcantilever with a good selectivity over other common metal ions.
Amino acids are required for activation of the mammalian target of rapamycin (mTOR) kinase which regulates protein translation, cell growth, and autophagy. Cell surface transporters that allow amino acids to enter the cell and signal to mTOR are unknown. We show that cellular uptake of L-glutamine and its subsequent rapid efflux in the presence of essential amino acids (EAA) is the rate-limiting step that activates mTOR. L-glutamine uptake is regulated by SLC1A5 and loss of SLC1A5 function inhibits cell growth and activates autophagy. The molecular basis for L-glutamine sensitivity is due to SLC7A5/SLC3A2, a bidirectional transporter that regulates the simultaneous efflux of L-glutamine out of cells and transport of L-leucine/EAA into cells. Certain tumor cell lines with high basal cellular levels of L-glutamine bypass the need for L-glutamine uptake and are primed for mTOR activation. Thus, L-glutamine flux regulates mTOR, translation and autophagy to coordinate cell growth and proliferation.
Chlorpromazine (CPZ)-induced morphological changes of living human cervical carcinoma cells were investigated by atomic force microscopy in near physiological condition. The results showed that the cell morphology changed visibly with time in the presence of CPZ (>21 microM). The cell membrane shrank gradually and detached finally from the substrate. After being treated with CPZ for 50 min, the cell volume increased by about 27.6% while its projective area (cell adhered to the substrate) decreased by about 12%. The mechanism was also discussed.
Preclinical data have shown the potential of the intraductal administration of chemotherapy for breast cancer prevention. Direct translation of this work has been stymied by the anatomical differences between rodents (one duct per teat) and women (5-9 ductal systems per breast). The objective of this phase I study was to show the safety and feasibility of intraductal administration of chemotherapy drugs into multiple ducts within one breast in women awaiting mastectomy for treatment of invasive cancer. Thirty subjects were enrolled in this dose escalation study conducted at a single center in Beijing, China. Under local anesthetic, one of two chemotherapy drugs, carboplatin or pegylated liposomal doxorubicin (PLD), was administered into five to eight ducts at three dose levels. Pharmacokinetic analysis has shown that carboplatin was rapidly absorbed into the bloodstream, whereas PLD, though more erratic, was absorbed after a delay. Pathologic analysis showed marked effects on breast duct epithelium in ducts treated with either drug compared with untreated ducts. The study investigators had no difficulty in identifying or cannulating ducts except in one case with a central cancer with subareolar involvement. This study shows the safety and feasibility of intraductal administration of chemotherapy into multiple ducts for the purpose of breast cancer prevention. This is an important step toward implementation of this strategy as a "chemical mastectomy", where the potential for carcinogenesis in the ductal epithelium is eliminated pharmacologically, locally, and without the need for surgery.
Mussels have been shown to attach to virtually all types of inorganic and organic surfaces via their adhesive proteins. The adhesive proteins secreted by mussels contain high concentrations of catechol and amine functional groups, which have similar functional groups with polydopamine (PDA). Inspired by mussels, a mild and environmentally friendly method was used to synthesize Ag nanoparticles (Ag NPs) on functionalized PDA-graphene nanosheets (PDA-GNS) with uniform and high dispersion. First, a uniform layer of PDA was coated on graphene oxide (GO) by polymerizing dopamine (DA) at room temperature. During the process GO was reduced by the DA. The PDA layer on the surface of GNS can be used as a nanoscale guide to form uniform Ag NPs on the surface of PDA-GNS. The obtained Ag-PDA-GNS hybrid materials are characterized by atomic force microscopy, transmission electron microscopy, UV-vis spectroscopy, Raman spectroscopy, X-ray photo-electron spectroscopy, X-ray diffraction, and thermal gravimetric analysis. The resultant Ag-PDA-GNS hybrid materials exhibited strong antibacterial properties to both Gram-negative and Gram-positive bacteria due to the synergistic effect of GNS and Ag NPs.
The role of cyclin B1 in the clinical therapeutic sensitivity of human esophageal squamous cell carcinoma (ESCC) remains to be defined. In this study, we found that elevated cyclin B1 expression attenuated the apoptosis induced by cisplatin or paclitaxel, while knockdown of cyclin B1 enhanced cisplatin or paclitaxel sensitivity in ESCC cells. Cyclin B1-mediated apoptosis may rely on the Bcl-2-dependent mitochondria-regulated intrinsic death pathway, and the antagonizing effect of cyclin B1 on chemotherapeutic agent-induced apoptosis was through PTEN/Akt pathway. Therefore, cyclin B1 might be a therapeutic target for the development of specific and efficient approaches in the treatment of ESCC.
The luteinizing hormone-releasing hormone- Pseudomonas aeruginosa exotoxin 40 (LHRH-PE40), is a candidate target drug associated with elevated LHRH receptor (LHRH-R) expression in malignant tumor tissue. The capability of LHRH-PE40 to recognize LHRH-Rs on a living cell membrane was studied with single molecular recognition force spectroscopy (SMFS) based on atomic force microscopy (AFM). The recognition force of LHRH-PE40/LHRH-R was compared with that of LHRH/LHRH-R by dynamic force spectroscopy. Meanwhile, cell growth inhibition assay and fluorescence imaging were presented as complementary characterization. The results show that LHRH moiety keeps its capability to recognize LHRH-R specifically, which implies that recombinant protein LHRH-PE40 can be a promising target drug.
Scatter me: A fast and cost-effective approach for the fabrication of surface-enhanced Raman scattering (SERS) arrays is developed. The method applied combines microcontact printing, electrodeposition, and galvanic replacement without the need for expensive instruments and intricate processing. The as-prepared arrays show excellent SERS activity and high reproducibility for Rhodamine 6G.
The self-assembled structures possess superior stability, biocompatibility and mechanical strength, and their study can provide insight into the use of creating novel biomaterials. Atomic force microscopy (AFM) images of single-stranded DNA (ssDNA) nanostructures show that well-ordered organization, high homogeneity, and molecular dimensions fractal-shaped fibers formed on a gold substrate covered with self-assembled monolayers (SAMs) of 1-hexadecanethiol (HDT). The nanoscaled architectures of ssDNA on HDT/Au changed remarkably following the process of diffusion-limited cluster aggregation (DLA) over time. The ssDNA fibers prefer to form on hydrophobic SAMs instead of hydrophilic SAMs, and the ssDNA has to have complementary regions in their sequences. This method might not be used only for the construction of fractal patterns, but also for the design and fabrication of functional DNA-based, self-assembled materials that exhibit self-similarity at multiple length scales.
In this article, we propose a facile one-pot solvothermal route for synthesizing TiO(2)-graphene composite nanosheets (TGCN). In the system, ethylene glycol not only as a reducing agent can convert graphene oxide to reduced graphene oxide nanosheets, but also is employed to control the hydrolysis and condensation rates of tetrabutoxytitanium. The obtained TGCN hybrid materials are characterized by atomic force microscopy, transmission electron microscopy, UV-vis spectroscopy, Raman spectroscopy, X-ray photo-electron spectroscopy, X-ray diffraction, and thermal gravimetric analysis. It is found that the quantity of H(2)O used in the reaction is the key to obtain high-quality product. The photocatalytic activities of the products are evaluated using the photocatalytic degradation of methylene blue (MB) as a probe reaction. The results showed that the obtained TGCN have an enhanced adsorption capacity and remarkable improvements in the photodegradation rate of MB under visible light compared to P25.
Fluorescent nanodiamonds (FNDs) have drawn much attention in recent years for biomedical imaging applications due to their desired physical properties including excellent photostability, high biocompatibility, extended far-red fluorescence emission, and ease of surface functionalization. Here we explore a new feature of FNDs, i.e. their photoacoustic emission capability, which may lead to potential applications of using FNDs as a dual imaging contrast agent for combined fluorescence and photoacoustic imaging modalities. We observed significant enhancement of photoacoustic emission from FNDs when they were conjugated with gold nanoparticles (GNPs).
A conundrum has long lingered over association of cytosol elongation factor Tu (EF-Tu) with bacterial surface. Here we investigated it with Acinetobacter baumannii, an emerging opportunistic pathogen associated with a wide spectrum of infectious diseases. The gene for A. baumannii EF-Tu was sequenced, and recombinant EF-Tu was purified for antibody development. EF-Tu on the bacterial surface and the outer membrane vesicles (OMVs) was revealed by immune electron microscopy, and its presence in the outer membrane (OM) and the OMV subproteomes was verified by Western blotting with the EF-Tu antibodies and confirmed by proteomic analyses. EF-Tu in the OM and the OMV subproteomes bound to fibronectin as detected by Western blot and confirmed by a label-free real-time optical sensor. The sensor that originates from photonic crystal structure in a total-Internal-reflection (PC-TIR) configuration was functionalized with fibronectin for characterizing EF-Tu binding. Altogether, with a novel combination of immunological, proteomical, and biophysical assays, these results suggest association of A. baumannii EF-Tu with the bacterial cell surface, OMVs, and fibronectin.
The Wnt signaling pathway is critical to the regulation of key cellular processes. When deregulated, it has been shown to play a crucial role in the growth and progression of multiple human cancers. The identification of small molecule modulators of Wnt signaling has proven challenging, largely due to the relative paucity of druggable nodes in this pathway. Several recent publications have identified small molecule inhibitors of the Wnt pathway, and tankyrase (TNKS) inhibition has been demonstrated to antagonize Wnt signaling via axin stabilization. Herein, we report the early hit assessment of a series of compounds previously reported to antagonize Wnt signaling. We report the biophysical, computational characterization, structure-activity relationship, and physicochemical properties of a novel series of [1,2,4]triazol-3-ylsulfanylmethyl)-3-phenyl-[1,2,4]oxadiazole inhibitors of TNKS1 and 2. Furthermore, a cocrystal structure of compound 24 complexed to TNKS1 demonstrates an alternate binding mode for PARP family member proteins that does not involve interactions with the nicotinamide binding pocket.
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