Purinergic signaling through the P2Y2 receptor regulates osteocytes' mechanosensitivity.

Osteocytes' response to dynamic loading plays a crucial role in regulating the bone mass but quickly becomes saturated such that downstream induction of bone formation plateaus. The underlying mechanisms that downregulate osteocytes' sensitivity and overall response to loading remain unknown. In other cell types, purinergic signaling through the P2Y2 receptor has the potential to downregulate the sensitivity to loading by modifying cell stiffness through actin polymerization and cytoskeleton organization. Herein, we examined the role of P2Y2 activation in regulating osteocytes' mechanotransduction using a P2Y2 knockout cell line alongside conditional knockout mice. Our findings demonstrate that the absence of P2Y2 expression in MLO-Y4 cells prevents actin polymerization while increasing the sensitivity to fluid flow-induced shear stress. Deleting osteocytes' P2Y2 expression in conditional-knockout mice enabled bone formation to increase when increasing the duration of exercise. Overall, P2Y2 activation under loading produces a negative feedback loop, limiting osteocytes' response to continuous loading by shifting the sensitivity to mechanical strain through actin stress fiber formation.

Mechanical evolution of metastatic cancer cells in three-dimensional microenvironment.

Cellular biomechanics plays critical roles in cancer metastasis and tumor progression. Existing studies on cancer cell biomechanics are mostly conducted in flat 2D conditions, where cells' behavior can differ considerably from those in 3D physiological environments. Despite great advances in developing 3D in vitro models, probing cellular elasticity in 3D conditions remains a major challenge for existing technologies. In this work, we utilize optical Brillouin microscopy to longitudinally acquire mechanical images of growing cancerous spheroids over the period of eight days. The dense mechanical mapping from Brillouin microscopy enables us to extract spatially resolved and temporally evolving mechanical features that were previously inaccessible. Using an established machine learning algorithm, we demonstrate that incorporating these extracted mechanical features significantly improves the classification accuracy of cancer cells, from 74% to 95%. Building on this finding, we have developed a deep learning pipeline capable of accurately differentiating cancerous spheroids from normal ones solely using Brillouin images, suggesting the mechanical features of cancer cells could potentially serve as a new biomarker in cancer classification and detection.

Potential application of nanoliposomes loaded with complex tannins from the seed shell of Euryale ferox in the anti-browning of fresh-cut asparagus lettuce.

Surface browning of plant-derived fresh-cut products is mainly caused by conversion of the phenolic compounds into o-quinones under tyrosinase catalysis. In this study, the rarely reported complex tannins from Euryale ferox seed shell (ECTs) constituted by the units of 35.60% condensed tannins and 64.40% hydrolysable tannins were shown to suppress the activity of tyrosinase efficiently, supporting the exploitation of ECTs into novel anti-browning agents. However, the utilization of ECTs in food preservation is often restricted because of their chemical instability to external environment. Further fabrication of nanoliposomes loaded with ECTs (ECTs-NLs) herein was carried out to improve the stability of ECTs. DLS, TEM, FTIR, DSC and XRD confirmed that ECTs were encapsulated into nanoliposomes successfully, and ECTs-NLs appeared as vesicle-like spherical morphology with favorable encapsulation efficiency, uniform particle size distribution and negative zeta-potential. The resulting ECTs-NLs were relatively stable in the dark at 4 °C. Nanoliposomal encapsulation significantly enhanced ECTs stability, thus protecting inhibitory effect of ECTs against tyrosinase. Furthermore, anti-browning evaluation proved that ECTs-NLs had distinct advantages over free ECTs in alleviating surface browning of fresh-cut asparagus lettuces. These results suggested that nanoliposomes were effective in stabilizing ECTs and ECTs-NLs could be potentially applied to the fresh-cut food industry.

Monitoring the Mechanical Evolution of Tissue During Neural Tube Closure of Chick Embryo.

Neural tube closure (NTC) is a critical process during embryonic development. Failure in this process can lead to neural tube defects, causing congenital malformations or even mortality. NTC involves a series of mechanisms on genetic, molecular, and mechanical levels. While mechanical regulation has become an increasingly attractive topic in recent years, it remains largely unexplored due to the lack of suitable technology for conducting mechanical testing of 3D embryonic tissue in situ. In response, we have developed a protocol for quantifying the mechanical properties of chicken embryonic tissue in a non-contact and non-invasive manner. This is achieved by integrating a confocal Brillouin microscope with an on-stage incubation system. To probe tissue mechanics, a pre-cultured embryo is collected and transferred to an on-stage incubator for ex ovo culture. Simultaneously, the mechanical images of the neural plate tissue are acquired by the Brillouin microscope at different time points during development. This protocol includes detailed descriptions of sample preparation, the implementation of Brillouin microscopy experiments, and data post-processing and analysis. By following this protocol, researchers can study the mechanical evolution of embryonic tissue during development longitudinally.

A Novel Role of Connective Tissue Growth Factor in the Regulation of the Epithelial Phenotype.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is a biological process where epithelial cells lose their adhesive properties and gain invasive, metastatic, and mesenchymal properties. Maintaining the balance between the epithelial and mesenchymal stage is essential for tissue homeostasis. Many of the genes promoting mesenchymal transformation have been identified; however, our understanding of the genes responsible for maintaining the epithelial phenotype is limited. Our objective was to identify the genes responsible for maintaining the epithelial phenotype and inhibiting EMT. METHODS: RNA seq was performed using an vitro model of EMT. CTGF expression was determined via qPCR and Western blot analysis. The knockout of CTGF was completed using the CTGF sgRNA CRISPR/CAS9. The tumorigenic potential was determined using NCG mice. RESULTS: The knockout of CTGF in epithelial ovarian cancer cells leads to the acquisition of functional characteristics associated with the mesenchymal phenotype such as anoikis resistance, cytoskeleton remodeling, increased cell stiffness, and the acquisition of invasion and tumorigenic capacity. CONCLUSIONS: We identified CTGF is an important regulator of the epithelial phenotype, and its loss is associated with the early cellular modifications required for EMT. We describe a novel role for CTGF, regulating cytoskeleton and the extracellular matrix interactions necessary for the conservation of epithelial structure and function. These findings provide a new window into understanding the early stages of mesenchymal transformation.

Versatile multimodal modality based on Brillouin light scattering and the photoacoustic effect.

Multimodal optical techniques are useful for the comprehensive characterization of material properties. In this work, we developed a new, to the best of our knowledge, multimodal technology that can simultaneously measure a subset of mechanical, optical, and acoustical properties of the sample and is based on the integration of Brillouin (Br) and photoacoustic (PA) microscopy. The proposed technique can acquire co-registered Br and PA signals from the sample. Importantly, using synergistic measurements of the speed of sound and Brillouin shift, the modality offers a new approach to quantifying the optical refractive index, which is a fundamental property of a material and is not accessible by either technique individually. As a proof of concept, we demonstrated the feasibility of integrating the two modalities and acquired the colocalized Br and time-resolved PA signals in a synthetic phantom made out of kerosene and CuSO4 aqueous solution. In addition, we measured the refractive index values of saline solutions and validated the result. Comparison with previously reported data showed a relative error of 0.3%. This further allowed us to directly quantify the longitudinal modulus of the sample with the colocalized Brillouin shift. While the scope of the current work is limited to introducing the combined Br-PA setup for the first time, we envision that this multimodal modality could open a new path for the multi-parametric analysis of material properties.

Non-contact and label-free biomechanical imaging: Stimulated Brillouin microscopy and beyond.

Brillouin microscopy based on spontaneous Brillouin scattering has emerged as a unique elastography technique because of its merit of non-contact, label-free, and high-resolution mechanical imaging of biological cell and tissue. Recently, several new optical modalities based on stimulated Brillouin scattering have been developed for biomechanical research. As the scattering efficiency of the stimulated process is much higher than its counterpart in the spontaneous process, stimulated Brillouin-based methods have the potential to significantly improve the speed and spectral resolution of existing Brillouin microscopy. Here, we review the ongoing technological advancements of three methods, including continuous wave stimulated Brillouin microscopy, impulsive stimulated Brillouin microscopy, and laser-induced picosecond ultrasonics. We describe the physical principle, the representative instrumentation, and biological application of each method. We further discuss the current limitations as well as the challenges for translating these methods into a visible biomedical instrument for biophysics and mechanobiology.

A versatile multimodal optical modality based on Brillouin light scattering and photoacoustic effect.

Multimodal optical imaging techniques are useful for various applications, including imaging biological samples for providing comprehensive material properties. In this work, we developed a new modality that can measure a set of mechanical, optical, and acoustical properties of a sample at microscopic resolution, which is based on the integration of Brillouin (Br) and photoacoustic (PA) microscopy. The proposed multimodal imaging technique not only can acquire co-registered Br and PA signals but also allows us to utilize the sound speed measured by PA to quantify the sample’s refractive index, which is a fundamental property of the material and cannot be measured by either technique individually. We demonstrated the colocalization of Br and time-resolved PA signals in a synthetic phantom made of kerosene and CuSO 4 aqueous solution. In addition, we measured the refractive index of saline solutions and validated the result against published data with a relative error of 0.3 %. This multimodal Br-PA modality could open a new way for characterizing biological samples in physiological and pathological conditions.

Exploring influences on the intention of motorcyclists to drink and ride: An investigation in a fourth-tier city of China.

OBJECTIVE: The analysis of motorcyclists' intention to drink and ride can help reduce the possibility of accidents caused by the relevant behavior of motorcyclists. The main objectives of this study are to identify important factors in motorcyclists' intention to drink and ride and to make some recommendations that could effectively reduce their riding intention after drinking. METHODS: To explore the effects of demographic and psychological variables on motorcyclists' behavioral intention to drink and ride, a questionnaire based on the extended theory of planned behavior was designed. Two hundred and five fully completed questionnaires were collected through a survey in Shaoguan, China. A hierarchical regression model was used to analyze observed data. RESULTS: The final hierarchical regression model explained 37.5% of the variance in intention to drink and ride. While initial tests showed that gender, marital status and age influenced some TPB variables, gender was the only demographic variable found to be significant on the riding intention after drinking alcohol in the hierarchical regression analysis. Among the psychological factors quantified by the extended theory of planned behavior, all factors had significant effects on intention except for risk perception, and subjective norms were the most influential factor. CONCLUSIONS: The extended theory of planned behavior can be well used to examine the factors that influence motorcyclists' intention to drink and ride. A more nuanced understanding of these factors can be found in the results.

Clinical characteristics and risk factors of cerebral cavernous malformation-related epilepsy.

PURPOSE: This study aimed to summarize the clinical characteristics and explore the risk factors for cerebral cavernous malformation (CCM)-related epilepsy (CRE). METHODS: We retrospectively analyzed the clinical data of patients with CCM in our cerebral vascular malformations database. Descriptive statistics were used to present the clinical characteristics of CRE patients. Patients were divided into a CRE and a non-CRE group according to clinical presentation. Binary logistic regression analysis was used to analyze the risk factors of CRE. RESULTS: A total of 199 patients with CCM confirmed by postoperative pathological examination were enrolled, 93 of whom were diagnosed with CRE, and 34 patients had drug-resistant epilepsy. The most common seizure type of CRE patients was focal to bilateral tonic-clonic seizure (FBTCS), followed by focal impaired awareness motor seizure. All CCM lesions were supratentorial, 97.8% of which involved the cerebral cortex, 86.0% of lesions had hemosiderin rim, and 50.5% of lesions were located in the temporal lobe. Binary logistic regression analysis indicated that CCM diagnosis age ≤ 44 years (odds ratio [OR] 2.79, p = 0.010), temporal lobe lesion location (OR = 9.07, p = 0.042), medial temporal lobe lesion (OR = 14.09, p = 0.002), cortical involvement of the lesion (OR = 32.77, p = 0.010), and hemosiderin rim around the lesion (OR = 16.48, p = 0.001) significantly increased the risk of CRE. CONCLUSIONS: The most common seizure type of CRE was FBTCS. Those whose CCM diagnosis age was ≤ 44 years, having a temporal lobe lesion location, especially the medial temporal lobe lesion, cortical involvement, and hemosiderin rim around the lesion had a higher risk of developing CRE.

Terahertz Spectroscopy for Accurate Identification of Panax quinquefolium Basing on Nonconjugated 24(R)-Pseudoginsenoside F11.

Panax quinquefolium is a perennial herbaceous plant that contains many beneficial ginsenosides with diverse pharmacological effects. 24(R)-pseudoginsenoside F11 is specific to P. quinquefolium, a useful biomarker for distinguishing this species from other related plants. However, because of its nonconjugated property and the complexity of existing detection methods, this biomarker cannot be used as the identification standard. We herein present a stable 24(R)-pseudoginsenoside F11 fingerprint spectrum in the terahertz band, thereby proving that F11 can be detected and quantitatively analyzed via terahertz spectroscopy. We also analyzed the sample by high-performance liquid chromatography-triple quadrupole mass spectrometry. The difference between the normalized data for the two analytical methods was less than 5%. Furthermore, P. quinquefolium from different areas and other substances can be clearly distinguished based on these terahertz spectra with a standard principal component analysis. Our method is a fast, simple, and cost-effective approach for identifying and quantitatively analyzing P. quinquefolium.

Molecular methylation detection based on terahertz metamaterial technology.

Terahertz wave has a good ability to identify biomolecules due to its fingerprint spectrum characteristics. However, the minimum detectable limit of terahertz technology by the conventional tablet pressing method is on the order of milligrams, which cannot meet the application requirements of low concentration detection in the biomedical field-near or below micrograms. Here, we proposed a method to enhance the detection sensitivity by designing a metamaterial chip with the absorption-induced transparency (AIT) effect, which can enhance the interaction between terahertz waves and biomolecules and lower the detectable limit. Taking 7-methylguanine (7-MG) as an example, based on its terahertz characteristic absorption peak, we designed a split-ring resonator (SRR) metamaterial chip, which has the advantages of high sensitivity, unlabeled detection, fast response and simple measurement. Its quantitative detection limit can reach 6.30 µg, which is about 500 times smaller than that of the traditional tablet pressing method (2.95 mg). In addition, for methylated and unmethylated substances, the chip exhibits different frequency shifts, which also realizes the qualitative identification effectively. These results provide a reference for the rapid and accurate diagnosis of diseases associated with molecular methylation in clinical medicine.

Terahertz Imaging and Spectroscopy in Cancer Diagnostics: A Technical Review.

Terahertz (THz) waves are electromagnetic waves with frequency in the range from 0.1 to 10 THz. THz waves have great potential in the biomedical field, especially in cancer diagnosis, because they exhibit low ionization energy and can be used to discern most biomolecules based on their spectral fingerprints. In this paper, we review the recent progress in two applications of THz waves in cancer diagnosis: imaging and spectroscopy. THz imaging is expected to help researchers and doctors attain a direct intuitive understanding of a cancerous area. THz spectroscopy is an efficient tool for component analysis of tissue samples to identify cancer biomarkers. Additionally, the advantages and disadvantages of the developed technologies for cancer diagnosis are discussed. Furthermore, auxiliary techniques that have been used to enhance the spectral signal-to-noise ratio (SNR) are also reviewed.

Exploring the effect of hydrophilic and hydrophobic structure of grafted polymeric micelles on drug loading.

The objective of this paper is to explore the effect of hydrophilic and hydrophobic structure of grafted polymeric micelles on drug loading, and elucidate whether drug-polymer compatibility, as predicted by Hansen solubility parameters (HSPs), can be used as a tool for drug-polymer pairs screening and guide the design of grafted polymeric micelles. HSPs of 27 drugs and three grafted copolymers were calculated according to group contribution method. The drug-polymer compatibilities were evaluated using the approaches of Flory-Huggins interaction parameters (χFH) and polarity difference (△Xp). Two models, model A and B, were put forward for drug-polymer compatibility prediction. In model A, hydrophilic/hydrophobic part as a whole was regarded as one segment. And, in model B, hydrophilic and hydrophobic segments were evaluated individually. First of all, using chitosan (CS)-grafted-glyceryl monooeate (GMO) based micelle as an example, the suitability of model A and model B for predicating drug-polymer compatibility was evaluated theoretically. Thereafter, corresponding experiments were carried out to check the validity of the theoretical prediction. It was demonstrated that Model B, which evaluates drug compatibility with both hydrophilic and hydrophobic segments of the copolymer, is more reliable for drug-polymer compatibility prediction. Moreover, the approach of model B allows for the selection of a defined grafted polymer with for a specific drug and vice versa. Thus, drug compatibility evaluation via HSPs with both hydrophilic and hydrophobic segments is a suitable tool for the rational design of grafted polymeric micelles. The molecular dynamics (MD) simulation study provided further support to the established model and experimental results.

Nanomicelle Based Peroral Delivery System for Enhanced Absorption and Sustained Release of 10-Hydrocamptothecin.

An effective sustained-release peroral drug delivery system is needed for chemotherapy. Here, we show that such a system can be achieved by designing polymeric nanomicelles combining mucoadhesion, enhanced absorption and controlled release. Chitosan and glyceryl-monooleate have many desirable properties, so we synthesized a novel chitosan derivative, chitosan-conjugated glyceryl monooleate. We loaded 10-hydroxycamptothecin (HCPT) into the cores of nanomicelles by pH-coacervation, which significantly improved drug loading and stability. We studied the pharmacokinetics of these drug-loaded nanomicelles, and they demonstrated remarkably prolonged circulation time in vivo up to 72 h. Orally administered HCPT-loaded nanomicelles also showed comparable antitumor effects and smaller changes in body weight compared to HCPT administered by injection. Most importantly, by using in vivo pharmacokinetic and pharmacodynamic studies, we showed that comparable antitumor effects can be achieved by peroral administration of HCPT-loaded nanomicelles every three days, and that the nanomicelles had less severe side effects. In vivo imaging provided direct evidence that the micelles were absorbed and exhibited sustained release after oral administration. These results indicate a promising future for nanomicelle-based peroral drug delivery as a superior alternative to injection, and they also provide guiding principles for designing amphiphilic copolymers.

Designing micellar nanocarriers with improved drug loading and stability based on solubility parameter.

The objective of this study is to demonstrate the feasibility of using solubility parameter as guidance for the design and identification of a stable micellar system with a high drug loading capacity for oral drug delivery. Using hydroxycamptothecin (HCPT) as a model drug, the effect of three hydrophobic blocks (fatty glycerides) grafted onto chitosan on the drug loading and stability of HCPT-loaded micellar nanoparticles formed by pH precipitation method were studied systematically. The Flory-Huggins interaction parameter (χFH) calculated by the group contribution method (GCM) and molecular dynamics simulation (MDS) was used to assess the compatibility between HCPT and the copolymers. The predicted order of compatibility between three chitosan derivatives and HCPT was verified experimentally. A high drug loading and remarkably stable micellar system for oral administration based on succinylated glycerol monooleate-chitosan was discovered in this study. Our study suggests that the miscibility between drug and copolymer is crucial to drug loading and stability of the micellar system. Thus, the calculation of χFH using GCM and MDS methods is useful for guiding the design or screening of a suitable copolymer for preparing drug-loaded micellar nanocarrier systems.

Uptake, transport and peroral absorption of fatty glyceride grafted chitosan copolymer-enoxaparin nanocomplexes: influence of glyceride chain length.

The objective of this paper is to elucidate the influence of fatty glyceride chain length in chitosan copolymers on the peroral absorption of enoxaparin. First of all, a series of chitosan copolymers with glyceryl monocaprylate (GM8), glyceryl monolaurate (GM12) and glyceryl monostearate (GM18) as the hydrophobic part were synthesized. The structure of the copolymers was characterized using proton nuclear magnetic resonance. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay demonstrated that all the copolymers were non-toxic. Enoxaparin nanocomplexes were prepared by self-assembly. Mucoadhesion of the nanocomplexes was characterized using the mucin particle method. Nanocomplex uptake and transport were quantified in Caco-2 cells and cellular localization was visualized by confocal laser scanning microscopy. Enoxaparin uptake was enhanced by nanocomplex formation, and was dependent on incubation time, concentration, temperature and glyceride chain length. The GM8 grafted chitosan-enoxaparin nanocomplex exhibited the strongest bioadhesion and the best uptake and transport in both cell culture and in vivo absorption in rats. The uptake mechanism was assumed to be adsorptive endocytosis via clathrin- and caveolae-mediated processes. In conclusion, oral absorption of enoxaparin can be further enhanced by using GM8 grafted chitosan copolymer as the carrier to form nanocomplexes.

Self-assembling semiconducting polymers--rods and gels from electronic materials.

In an effort to favor the formation of straight polymer chains without crystalline grain boundaries, we have synthesized an amphiphilic conjugated polyelectrolyte, poly(fluorene-alt-thiophene) (PFT), which self-assembles in aqueous solutions to form cylindrical micelles. In contrast to many diblock copolymer assemblies, the semiconducting backbone runs parallel, not perpendicular, to the long axis of the cylindrical micelle. Solution-phase micelle formation is observed by X-ray and visible light scattering. The micelles can be cast as thin films, and the cylindrical morphology is preserved in the solid state. The effects of self-assembly are also observed through spectral shifts in optical absorption and photoluminescence. Solutions of higher-molecular-weight PFT micelles form gel networks at sufficiently high aqueous concentrations. Rheological characterization of the PFT gels reveals solid-like behavior and strain hardening below the yield point, properties similar to those found in entangled gels formed from surfactant-based micelles. Finally, electrical measurements on diode test structures indicate that, despite a complete lack of crystallinity in these self-assembled polymers, they effectively conduct electricity.

Regioregular copolymers of 3-alkoxythiophene and their photovoltaic application.

Low band gap conjugated polymers with proper energy levels for charge transfer are required to achieve high-efficiency polymer solar cells. We report the synthesis and characterization of two new regioregular copolymers that are based on 3-alkoxythiophene monomers: poly(3-octylthiophene-2,5-diyl-co-3-decyloxythiophene-2,5-diyl) (POT-co-DOT) and poly{(9,9-dioctylfluorene)-2,7-diyl-alt-[4,7-bis(3-decyloxythien-2-yl)-2,1,3-benzothiadiazole]-5',5' '-diyl} (PF-co-DTB). Compared to the alkyl substituents, the alkoxy side chains on the thiophene units can effectively lower the band gap of copolymers and enhance the charge transfer to electron acceptors such as (6,6)-phenyl C(61)-butyric acid methyl ester (PCBM). The chemical structure and regioregularity of the copolymers were confirmed by NMR. Both copolymers are readily soluble in organic solvents and form high-quality thin films. Electrochemical and photophysical studies reveal band gaps of 1.64 eV for POT-co-DOT and 1.78 eV for PF-co-DTB. Bulk heterojunction photovoltaic devices were fabricated using blends of these copolymers with PCBM as the active layer, ITO-glass as the anode, and aluminum as the cathode. Power conversion efficiency of 1.6% was obtained under simulated solar light AM 1.5 G (100 mW/cm(2)) from a solar cell with an active layer containing 20 wt % PF-co-DTB and 80 wt % PCBM. Regioregular poly(3-decyloxythiophene-2,5-diyl) (P3DOT) was also studied for comparison purposes.