Preparation, characterization, and anticancer effects of an inclusion complex of coixol with ß-cyclodextrin polymers.

CONTEXT: Coix [Coix lacryma-jobi L. var. mayuen (Roman.) Stapf (Poaceae)], a crop of medicinal and edible significance, contains coixol, which has demonstrated anticancer properties. However, the limited solubility of coixol restricts its potential therapeutic applications. OBJECTIVE: This study prepared a water-soluble coixol-ß-cyclodextrin polymer (CDP) inclusion compound and evaluated its anticancer effect. MATERIALS AND METHODS: The coixol-CDP compound was synthesized through a solvent-stirring and freeze-drying technique. Its coixol content was quantified using HPLC, and its stability was tested under various conditions. The anticancer effects of the coixol-CDP compound (4.129, 8.259, 16.518, and 33.035 mg/L for 24, 48, and 72 h) on the proliferation of non-small cell lung cancer (NSCLC) A549 cells were evaluated using an MTT assay; cell morphology was examined by Hoechst nuclear staining; apoptosis and cell cycle was detected by flow cytometry; and the expression of apoptosis-related proteins was assessed by Western blots. RESULTS: The water-soluble coixol-CDP inclusion compound was successfully prepared with an inclusion ratio of 86.6% and an inclusion yield rate of 84.1%. The coixol content of the compound was 5.63% and the compound remained stable under various conditions. Compared to coixol alone, all 24, 48, and 72 h administrations with the coixol-CDP compound exhibited lower IC50 values (33.93 ± 2.28, 16.80 ± 1.46, and 6.93 ± 0.83 mg/L) in A549 cells; the compound also showed stronger regulatory effects on apoptosis-related proteins. DISCUSSION AND CONCLUSIONS: These findings offer a new perspective for the potential clinical application of Coix in NSCLC therapy and its future research.

Airway dysbiosis accelerates lung function decline in chronic obstructive pulmonary disease.

Progressive lung function decline is a hallmark of chronic obstructive pulmonary disease (COPD). Airway dysbiosis occurs in COPD, but whether it contributes to disease progression remains unknown. Here, we show, through a longitudinal analysis of two cohorts involving four UK centers, that baseline airway dysbiosis in COPD patients, characterized by the enrichment of opportunistic pathogenic taxa, associates with a rapid forced expiratory volume in 1 s (FEV1) decline over 2 years. Dysbiosis associates with exacerbation-related FEV1 fall and sudden FEV1 fall at stability, contributing to long-term FEV1 decline. A third cohort in China further validates the microbiota-FEV1-decline association. Human multi-omics and murine studies show that airway Staphylococcus aureus colonization promotes lung function decline through homocysteine, which elicits a neutrophil apoptosis-to-NETosis shift via the AKT1-S100A8/A9 axis. S. aureus depletion via bacteriophages restores lung function in emphysema mice, providing a fresh approach to slow COPD progression by targeting the airway microbiome.

Liberation of daidzein by gut microbial ß-galactosidase suppresses acetaminophen-induced hepatotoxicity in mice.

Acetaminophen (APAP) overdose is a leading cause of drug-induced liver injury (DILI). The impact of the gut microbiota and associated metabolites on APAP and liver function remains unclear. We show that APAP disturbance is associated with a distinct gut microbial community, with notable decreases in Lactobacillus vaginalis. Mice receiving L. vaginalis showed resistance to APAP hepatotoxicity due to the liberation of the isoflavone daidzein from the diet by bacterial ß-galactosidase. The hepatoprotective effects of L. vaginalis in APAP-exposed germ-free mice were abolished with a ß-galactosidase inhibitor. Similarly, ß-galactosidase-deficient L. vaginalis produced poorer outcomes in APAP-treated mice than the wild-type strain, but these differences were overcome with daidzein administration. Mechanistically, daidzein prevented ferroptotic death, which was linked to decreased expression of farnesyl diphosphate synthase (Fdps) that activated a key ferroptosis pathway involving AKT-GSK3ß-Nrf2. Thus, liberation of daidzein by L. vaginalis ß-galactosidase inhibits Fdps-mediated hepatocyte ferroptosis, providing promising therapeutic approaches for DILI.

Pregnancy-induced changes to the gut microbiota drive macrophage pyroptosis and exacerbate septic inflammation.

The physiological and immune changes that occur during pregnancy are associated with worsened disease outcomes during infection and sepsis. How these perturbations exacerbate inflammation has not been explored. Here, using antibiotic treatment and fecal microbial transfers, we showed that sepsis susceptibility is driven by pregnancy-induced changes to gut microbiome in mice and humans. Integrative multiomics and genetically engineered bacteria revealed that reduced Parabacteroides merdae (P. merdae) abundance during pregnancy led to decreased formononetin (FMN) and increased macrophage death. Mechanistically, FMN inhibited macrophage pyroptosis by suppressing nuclear accumulation of hnRNPUL2 and subsequent binding to the Nlrp3 promoter. Treatment with FMN or deletion of murine hnRNPUL2 protected against septic inflammation. Intestinal abundances of P. merdae and FMN inversely correlated with the progression of septic patients. Our data reveal a microbe-immune axis that is disrupted in pregnant septic hosts, highlighting the potential of the FMN-hnRNPUL2-NLRP3 axis in providing promising therapeutic strategies for sepsis.

Multi-omics analyses of airway host-microbe interactions in chronic obstructive pulmonary disease identify potential therapeutic interventions.

The mechanistic role of the airway microbiome in chronic obstructive pulmonary disease (COPD) remains largely unexplored. We present a landscape of airway microbe-host interactions in COPD through an in-depth profiling of the sputum metagenome, metabolome, host transcriptome and proteome from 99 patients with COPD and 36 healthy individuals in China. Multi-omics data were integrated using sequential mediation analysis, to assess in silico associations of the microbiome with two primary COPD inflammatory endotypes, neutrophilic or eosinophilic inflammation, mediated through microbial metabolic interaction with host gene expression. Hypotheses of microbiome-metabolite-host interaction were identified by leveraging microbial genetic information and established metabolite-human gene pairs. A prominent hypothesis for neutrophil-predominant COPD was altered tryptophan metabolism in airway lactobacilli associated with reduced indole-3-acetic acid (IAA), which was in turn linked to perturbed host interleukin-22 signalling and epithelial cell apoptosis pathways. In vivo and in vitro studies showed that airway microbiome-derived IAA mitigates neutrophilic inflammation, apoptosis, emphysema and lung function decline, via macrophage-epithelial cell cross-talk mediated by interleukin-22. Intranasal inoculation of two airway lactobacilli restored IAA and recapitulated its protective effects in mice. These findings provide the rationale for therapeutically targeting microbe-host interaction in COPD.

Nanomedicines for Near-Infrared Fluorescent Lifetime-Based Bioimaging.

Nanomedicines refer to the application of nanotechnology in disease diagnosis, treatment, and monitoring. Bioimaging provides crucial biological information for disease diagnosis and treatment monitoring. Fluorescent bioimaging shows the advantages of good contrast and a vast variety of signal readouts and yet suffers from imaging depth due to the background noise from the autofluorescence of tissue and light scattering. Near-infrared fluorescent lifetime bioimaging (NIR- FLTB) suppresses such background noises and significantly improves signal-to-background ratio. This article gives an overview of recent advances in NIR- FLTB using organic compounds and nanomaterials as contrast agent (CA). The advantages and disadvantages of each CA are discussed in detail. We survey relevant reports about NIR-FLTB in recent years and summarize important findings or progresses. In addition, emerging hybrid bioimaging techniques are introduced, such as ultrasound-modulated FLTB. The challenges and an outlook for NIR- FLTB development are discussed at the end, aiming to provide references and inspire new ideas for future nanomedicine development.

Nanomedicines: A Potential Treatment for Blood Disorder Diseases.

Blood disorder diseases (BDDs), also known as hematologic, is one of the diseases owing to hematopoietic system disorder. Chemotherapy, bone marrow transplantation, and stem cells therapy have been used to treat BDDs. However, the cure rates are still low due to the availability of the right type of bone marrow and the likelihood of recurrence and infection. With the rapid development of nanotechnology in the field of biomedicine, artificial blood or blood substitute has shown promising features for the emergency treatment of BDDs. Herein, we surveyed recent advances in the development of artificial blood components: gas carrier components (erythrocyte substitutes), immune response components (white blood cell substitutes), and hemostasis-responsive components (platelet substitutes). Platelet-inspired nanomedicines for cancer treatment were also discussed. The challenges and prospects of these treatment options in future nanomedicine development are discussed.

Newly-Engineered Materials for Bio-Imaging Technology: A Focus on the Hybrid System of Ultrasound and Fluorescence.

As an emerging technique, ultrasound-modulated fluorescence (UMF), or ultrasound switchable fluorescence (USF) bioimaging has shown promising features to produce deep-tissue and high-resolution fluorescence imaging for biomedical research and health diagnosis. The success of UMF or USF heavily relies on the design of their contrast agents (CAs). We herein surveyed recent advances in the development of such unique CAs, including configuration, mechanism, stability, sensitivity, and selectivity. Meanwhile, UMF or USF instrumentation has emerged as developmental breakthrough technologies to existing bio-imaging techniques. The best performance of UMF or USF bio-imaging requires an interactive response between CAs and the instrument. In this review, the description of UMF or USF instrumentation are also included for clarification and better understanding. Finally, the UMF and USF's performance in bioimaging is evaluated based on signal-to-noise ratio, resolution, imaging depth and speed, using photoacoustic imaging (PAI) as a standard, a well-developed technique of hybrid bio-imaging. Unlike PAI, UMF or USF is still in its early stage. Although results demonstrated a proof-of-concept landmark being reached, significant efforts are needed to improve the performance of UMF or USF.

High-Resolution Ultrasound-Switchable Fluorescence Imaging in Centimeter-Deep Tissue Phantoms with High Signal-To-Noise Ratio and High Sensitivity via Novel Contrast Agents.

For many years, investigators have sought after high-resolution fluorescence imaging in centimeter-deep tissue because many interesting in vivo phenomena-such as the presence of immune system cells, tumor angiogenesis, and metastasis-may be located deep in tissue. Previously, we developed a new imaging technique to achieve high spatial resolution in sub-centimeter deep tissue phantoms named continuous-wave ultrasound-switchable fluorescence (CW-USF). The principle is to use a focused ultrasound wave to externally and locally switch on and off the fluorophore emission from a small volume (close to ultrasound focal volume). By making improvements in three aspects of this technique: excellent near-infrared USF contrast agents, a sensitive frequency-domain USF imaging system, and an effective signal processing algorithm, for the first time this study has achieved high spatial resolution (~ 900 µm) in 3-centimeter-deep tissue phantoms with high signal-to-noise ratio (SNR) and high sensitivity (3.4 picomoles of fluorophore in a volume of 68 nanoliters can be detected). We have achieved these results in both tissue-mimic phantoms and porcine muscle tissues. We have also demonstrated multi-color USF to image and distinguish two fluorophores with different wavelengths, which might be very useful for simultaneously imaging of multiple targets and observing their interactions in the future. This work has opened the door for future studies of high-resolution centimeter-deep tissue fluorescence imaging.

Ultrasound-modulated fluorescence based on donor-acceptor-labeled microbubbles.

A fluorescence resonance energy transfer (FRET)-based microbubble contrast agent system was designed to experimentally demonstrate the concept of ultrasound-modulated fluorescence (UMF). Microbubbles were simultaneously labeled with donor and acceptor fluorophores on the surface to minimize self-quenching and maximize FRET. In response to ultrasound, the quenching efficiency was greatly modulated by changing the distance between the donor and acceptor molecules through microbubble size oscillations. Both donors and acceptors exhibited UMF on individual microbubbles. The UMF strength of the donor was more significant compared to that of the acceptor. Furthermore, the UMF of the donor was observed from a microbubble solution in a turbid media. This study exploits the feasibility of donor­acceptor labeled microbubbles as UMF contrast agents.

Ultrasound-modulated fluorescence based on fluorescent microbubbles.

Ultrasound-modulated fluorescence (UMF) imaging has been proposed to provide fluorescent contrast while maintaining ultrasound resolution in an optical-scattering medium (such as biological tissue). The major challenge is to extract the weakly modulated fluorescent signal from a bright and unmodulated background. UMF was experimentally demonstrated based on fluorophore-labeled microbubble contrast agents. These contrast agents were produced by conjugating N-hydroxysuccinimide (NHS)-ester-attached fluorophores on the surface of amine-functionalized microbubbles. The fluorophore surface concentration was controlled so that a significant self-quenching effect occurred when no ultrasound was applied. The intensity of the fluorescent emission was modulated when microbubbles were oscillated by ultrasound pulses, presented as UMF signal. Our results demonstrated that the UMF signals were highly dependent on the microbubbles' oscillation amplitude and the initial surface fluorophore-quenching status. A maximum of ∼42% UMF modulation depth was achieved with a single microbubble under an ultrasound peak-to-peak pressure of 675 kPa. Further, UMF was detected from a 500-µm tube filled with contrast agents in water and scattering media with ultrasound resolution. These results indicate that ultrasound-modulated fluorescent microbubble contrast agents can potentially be used for fluorescence-based molecular imaging with ultrasound resolution in the future.

Re-evaluation of biotin-streptavidin conjugation in Förster resonance energy transfer applications.

Bioaffinity conjugation between streptavidin (SA) and biotin has been widely used to link donors and acceptors for investigating the distance-dependent Förster resonance energy transfer (FRET). When studying a commonly used FRET system of (QD-SA)-(biotin-DNA-dye) [donor: quantum dot (QD); acceptor: small organic fluorescent dye; and linker: deoxyribose nucleic acid (DNA) molecule via SA-biotin conjugation], however, a contradictory finding was recently reported in the literature. It was found that the FRET lost its dependence on the number of DNA base pairs when using a phosphate-buffered saline (PBS) solution. We found that the conflicted results were caused by the ionic strength of the adopted buffer solutions. Our results suggest that the dependent FRET on the number of DNA bases is favorable in a low-ionic-strength buffer, whereas in relatively high-ionic-strength buffers, the FRET loses the DNA length dependence. We propose that the independence is mainly caused by the conformational change of DNA molecules from a stretched to a coiled mode when the cations in the high-ionic-strength buffer neutralize the negatively charged backbone of DNA molecules, thereby bringing the acceptors close to the donors.

High resolution imaging beyond the acoustic diffraction limit in deep tissue via ultrasound-switchable NIR fluorescence.

Fluorescence imaging in deep tissue with high spatial resolution is highly desirable because it can provide details about tissue's structural, functional, and molecular information. Unfortunately, current fluorescence imaging techniques are limited either in penetration depth (microscopy) or spatial resolution (diffuse light based imaging) as a result of strong light scattering in deep tissue. To overcome this limitation, we developed an ultrasound-switchable fluorescence (USF) imaging technique whereby ultrasound was used to switch on/off the emission of near infrared (NIR) fluorophores. We synthesized and characterized unique NIR USF contrast agents. The excellent switching properties of these agents, combined with the sensitive USF imaging system developed in this study, enabled us to image fluorescent targets in deep tissue with spatial resolution beyond the acoustic diffraction limit.

Structure-dependent mitochondrial dysfunction and hypoxia induced with single-walled carbon nanotubes.

Cytotoxicity of nanomaterials on living systems is known to be affected by their size, shape, surface chemistry, and other physicochemical properties. Exposure to a well-characterized subpopulation of specific nanomaterials is therefore desired to reveal more detailed mechanisms. This study develops scalable density gradient ultracentrifugation sorting of highly dispersed single-walled carbon nanotubes (SWNTs) into four distinct bands based on diameter, aggregation, and structural integrity, with greatly improved efficiency, yield, and reproducibility. With guarantee of high yield and stability of four SWNT fractions, it is possible for the first time, to investigate the structure-dependent bioeffects of four SWNT fractions. it is possible Among these, singly-dispersed integral SWNTs show no significant effects on the mitochondrial functions and hypoxia. The aggregated integral SWNTs show more significant effects on the mitochondrial dysfunction and hypoxia compared to the aggregated SWNTs with poor structure integrity. Then, it is found that the aggregated integral SWNTs induced the irregular mitochondria respiratory and pro-apoptotic proteins activation, while aggregated SWNTs with poor structure integrity greatly enhanced reactive oxygen species (ROS) levels. This work supports the view that control of the distinct structure characteristics of SWNTs helps establish clearer structure-bioeffect correlation and health risk assessment. It is also hoped that these results can help in the design of nanomaterials with higher efficiency and accuracy in subcellular translocation.

Development of Ultrasound-switchable Fluorescence Imaging Contrast Agents based on Thermosensitive Polymers and Nanoparticles.

In this work we first introduced a recently developed high-resolution, deep-tissue imaging technique, ultrasound-switchable fluorescence (USF). The imaging principles based on two types of USF contrast agents were reviewed. To improve USF imaging techniques further, excellent USF contrast agents were developed based on high-performance thermoresponsive polymers and environment-sensitive fluorophores. Herein, such contrast agents were synthesized and characterized with five key parameters: (1) peak excitation and emission wavelengths (λex and λem), (2) the fluorescence intensity ratio between on and off states (IOn/IOff), (3) the fluorescence lifetime ratio between on and off states (τOn/τOff), (4) the temperature threshold to switch on fluorophores (Tth), and (5) the temperature transition bandwidth (TBW). We mainly investigated fluorescence intensity and lifetime changes of four environment-sensitive dyes [7-(2-Aminoethylamino)-N,N-dimethyl-4-benzofurazansulfonamide (DBD-ED), St633, Sq660, and St700] as a function of temperature, while the dye was attached to poly(N-isopropylacrylamide) linear polymers or encapsulated in nanoparticles. Six fluorescence resonance energy transfer systems were invented in which both the donor (DBD-ED or ST425) and the acceptor (Sq660) were adopted. Our results indicate that three Förster resonance energy transfer systems, where both IOn/IOff and τOn/τOff are larger than 2.5, are promising for application in future surface tissue bioimaging by USF technique.

Rapid and efficient sonochemical formation of gold nanoparticles under ambient conditions using functional alkoxysilane.

Gold nanoparticles (NPs) are rapidly and efficiently formed under ambient conditions with a novel and highly-efficient sonochemical promoter. Despite of the presence of free oxygen, 3-glycidoxypropyltrimethoxysilane (GPTMS) showed remarkable efficiency in promoting the reduction rate of Au (III) than that of conventional promoters (primary alcohols). This is likely attributed to the formation of a variety of radical scavengers, which are alcoholic products from sonochemical hydrolysis of the epoxide group and methoxysilane moieties of GPTMS under weakly acidic conditions. Interestingly, the promotion is quenched by amine- or thiol-functionalized alkoxysilane, thereby producing marginal amounts of gold NPs. Furthermore, products of hydrolyzed GPTMS were confirmed to attach on the surface of gold NPs by attenuated total reflectance-Fourier transform infrared spectroscopy. However, according to transmission electron microscopy images, gold NPs that were produced in the presence of GPTMS tend to fuse with each other as condensation of silanols occurs, forming worm- or nugget-like gold nanostructures. The use of long chain surfactants (i.e. polyethylene glycol terminated with hydroxyl or carboxyl) inhibited the fusion, leading to mono-dispersed gold NPs. Additionally, the fact that this approach requires neither an ultrasound source with high frequency nor anaerobic conditions provides a huge advantage. These findings could potentially open an avenue for rapid and large-scale green-synthesis of gold NPs in future work.

A visualized observation of calcium-dependent gelsolin activity upon the surface coverage of fluorescent-tagged actin filaments.

Gelsolin regulates the dynamics of F-actin by binding to F-actin to sever and cap. In the present study, a novel approach is introduced to observe gelsolin activity through the coverage of surface-bound F-actin. Gelsolin was immobilized on streptavidin coated surface using biotinylation and, as a result, the interaction between gelsolin and F-actin was visualized. Consequently, the coverage of F-actin reflects the activity of gelsolin as a function of free Ca(2+) concentrations. In order to prevent non-specific binding of F-actin, the combinations of BSA and Tween-20 as blocking agents were investigated. Moreover, the measurement of the length of F-actin with actin-gelsolin mixtures at various ratios provided the verification of gelsolin activity after biotinylation. The data shows the increase in Ca(2+) concentration leads to a proportional increase in F-actin coverage, giving to half-maximal coverage at ~2.9 µM. Furthermore, the length of bound F-actin was found to decrease along with increasing Ca(2+) concentration, and full-length F-actin was rarely observed. This may suggest that severing and capping activities of gelsolin occur without more additional Ca(2+) for subsequent activation after full-length gelsolin binds to a side of F-actin. This finding may provide a key to understand gelsolin activity.

Label-free electrochemical measurement of protein tyrosine kinase activity and inhibition based on electro-catalyzed tyrosine signaling.

A novel label-free electrochemical method for measuring the activity of protein tyrosine kinases (PTK) has been developed. Epidermal growth factor receptor (EGFR), a typical PTK associated with a large percentage of all solid tumors, was used as the model kinase. Poly(glu, tyr) (4:1) peptide, as a substrate of EGFR, was covalently immobilized on the surface of indium tin oxide (ITO) electrode by silane chemistry. The tyrosine (Tyr) residue in the polypeptide served as an electrochemical signal reporter. Its voltammetric current was catalyzed by a dissolved electron mediator Os(bpy)(3)(2+) (bpy=2,2'-bipyridine) for increased sensitivity. Phosphorylation of the Tyr led to a loss of its electrochemical current, thus providing a sensing mechanism for PTK activity. Experimental conditions for the silanization of ITO surface and immobilization of polypeptide were investigated in details to facilitate the generation of Tyr electrochemical signal. The proposed biosensor exhibited high sensitivity and excellent stability. The limit of detection for EGFR was 1 UmL(-1). Furthermore, this biosensor can also be used for quantitative analysis of kinase inhibition. On the basis of the inhibitor concentration dependent electrochemical signal, the half-maximal inhibition value IC(50) of three EGFR inhibitors, PD-153035, OSI-774 and ZD-1839, and their corresponding inhibition constants K(i) were estimated, which were in agreement with those obtained from the conventional kinase assay. This electrochemical biosensor can be implemented in an array format for the high throughput assay of in vitro PTK activity and PTK inhibitors screening for practical diagnostic application and drug discovery.

Selective attachment of F-actin with controlled length for developing an intelligent nanodevice.

Development of the nanodevice that myosin-coated beads "walk" on actin filaments (F-actin) tracks for in vitro nanotransportation was hindered due to the difficulty of assembling large-area well-orientated F-actin tracks on the surface. In this work, we present a selective attachment of F-actin with controlled length on a patterned surface by employing biotinylated capped protein gelsolin as intermediate anchoring bridge. A patterned streptavidin layer was formed via coupling with a biotin layer that photo-actively attached to an amine-functionalized glass surface. The patterned film was found stable and homogenous compared to that obtained by microcontact printing method, according to the profiling with fluorescence microscopy. By a secondary blocking process, non-specific binding of F-actin to the patterned surface through electrostatic adsorption can be resisted. The length variation of F-actin as a function of gelsolin concentration was also investigated, implying that F-actin is appropriately of 2.5 µm in average length once F-actin/gelsolin molar ratio is 4:1. Finally, the selective attachment of F-actin was well characterized with quantifying the number of attached F-actin per unit area in the patterned areas over that in blocked areas. The density of F-actin was estimated at c.a. 2 µm(2) per actin filament molecule so that the distance between one another actin filament is estimated as c.a. 1.41-1.97 µm. The unique properties of F-actin, e.g. well flexibility or electrical conductivity, make it feasible to lay them down and form unidirectional aligned tracks by fluidic flow or electrical field. This may open a possibility for the long-distant movement of myosin-coated beads, offering a novel discipline for the development of micro-biochip in vitro.