Organic Nanoparticles Based on D-A-D Small Molecule: Self-Assembly, Photophysical Properties, and Synergistic Photodynamic/Photothermal Effects

Cancer is one of the major diseases threatening human health. Traditional cancer treatments have notable side-effects as they can damage the immune system. Recently, phototherapy, as a potential strategy for clinical cancer therapy, has received wide attention due to its minimal invasiveness and high efficiency. Herein, a small organic molecule (PTA) with a D-A-D structure was prepared via a Sonogashira coupling reaction between the electron-withdrawing dibromo-perylenediimide and electron-donating 4-ethynyl-N,N-diphenylaniline. The amphiphilic organic molecule was then transformed into nanoparticles (PTA-NPs) through the self-assembling method. Upon laser irradiation at 635 nm, PTA-NPs displayed a high photothermal conversion efficiency (PCE = 43%) together with efficient reactive oxygen species (ROS) generation. The fluorescence images also indicated the production of ROS in cancer cells with PTA-NPs. In addition, the biocompatibility and photocytotoxicity of PTA-NPs were evaluated by 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and live/dead cell co-staining test. Therefore, the as-prepared organic nanomaterials were demonstrated as promising nanomaterials for cancer phototherapy in the clinic.

Regulatory Effect of Irresistin-16 on Competitive Dual-Species Biofilms Composed of Streptococcus mutans and Streptococcus sanguinis

Based on the ecological plaque hypothesis, suppressing opportunistic pathogens within biofilms, rather than killing microbes indiscriminately, could be a biofilm control strategy for managing dental caries. The present study aimed to evaluate the effects of irresistin-16 (IRS-16) on competitive dual-species biofilms, which consisted of the conditional cariogenic agent Streptococcus mutans (S. mutans) and oral commensal bacteria Streptococcus sanguinis (S. sanguinis). Bacterial growth and biofilm formation were monitored using growth curve and crystal violet staining, respectively. The microbial proportion was determined using fluorescence in situ hybridization. A 2, 5-diphenyltetrazolium bromide assay was used to measure the metabolic activity of biofilms. Bacterial/extracellular polysaccharide (EPS) dyeing, together with water-insoluble EPS measurements, were used to estimate EPS synthesis. A lactic acid assay was performed to detect lactic acid generation in biofilms. The cytotoxicity of IRS-16 was evaluated in mouse fibroblast L929 cells using a live/dead cell viability assay and cell counting kit-8 assay. Our results showed that IRS-16 exhibited selective anti-biofilm activity, leading to a remarkable survival disadvantage of S. mutans within competitive dual-species biofilms. In addition, the metabolic activity, EPS synthesis, and acid generation of dual-species biofilms were significantly reduced by IRS-16. Moreover, IRS-16 showed minimal cytotoxicity against mouse fibroblast L929 cells. In conclusion, IRS-16 exhibited remarkable regulatory effects on dual-species biofilms composed of S. mutans and S. sanguinis with low cytotoxicity, suggesting that it may have potential for use in caries management through ecological biofilm control.

Egg White Alginate as a Novel Scaffold Biomaterial for 3D Salivary Cell Culturing

Saliva production by salivary glands play a crucial role in oral health. The loss of salivary gland function could lead to xerostomia, a condition also known as dry mouth. Significant reduction in saliva production could lead to further complications such as difficulty in speech, mastication, and increased susceptibility to dental caries and oral infections and diseases. While some palliative treatments are available for xerostomia, there are no curative treatments to date. This study explores the use of Egg White Alginate (EWA), as an alternative scaffold to Matrigel® for culturing 3D salivary gland cells. A protocol for an optimized EWA was established by comparing cell viability using 1%, 2%, and 3% alginate solution. The normal salivary simian virus 40-immortalized acinar cell (NS-SV-AC) and the submandibular gland-human-1 (SMG-hu-1) cell lines were also used to compare the spheroid formation and cell viability properties of both scaffold biomaterials; cell viability was observed over 10 days using a Live–Dead Cell Assay. Cell viability and spheroid size in 2% EWA was significantly greater than 1% and 3%. It is evident that EWA can support salivary cell survivability as well as form larger spheroids when compared to cells grown in Matrigel®. However, further investigations are necessary as it is unclear if cultured cells were proliferating or aggregating.

Experimental Validation of Stability and Applicability of Start Growth Time Method For High-Throughput Bacterial Ecotoxicity Assessment.

Ecotoxicity assessments based on bacteria as model organisms is widely used for routine toxicity screening because it has advantages of time-saving, high sensitivity, cost-effectiveness, and less ethical responsibility. Determination of ecotoxicity effect via bacterial growth can avoid the restriction of model bacteria selection and unique equipment requirement, but traditional viable cell count methods are relatively labor- and time-intensive. The Start Growth Method (SGT) is a high-throughput and time-conserving method to determine the amount of viable bacterial cells. However, its usability and stability for ecotoxicity assessment are rarely studied. This study confirmed its applicability in terms of bacterial types (gram-positive and gram-negative), growth phases (middle exponential and early stationary phases), and simultaneous existence of dead cells (adjustment by flow cytometry). Our results verified that the stability of establishing SGT correlation is independent of the bacterial type and dead-cell portion. Moreover, we only observed the effect of growth phases on the slope value of established SGT correlation in Shewanella oneidensis, which suggests that preparing inoculum for the SGT method should be consistent in keeping its stability. Our results also elucidate that the SGT values and the live cell percentages meet the non-linear exponential correlation with high correlation coefficients from 0.97 to 0.99 for all the examined bacteria. The non-linear exponential correlation facilitates the application of the SGT method on the ecotoxicity assessment. Finally, applying the exponential SGT correlation to evaluate the ecotoxicity effect of copper ions on E. coli was experimentally validated. The SGT-based method would require about 6 to 7 hours to finish the assessment and obtained an estimated EC50 at 2.27 ± 0.04 mM. This study demonstrates that the exponential SGT correlation can be a high-throughput, time-conversing, and wide-applicable method for bacterial ecotoxicity assessment.

In Vivo Evaluation of Gallium-68-Labeled IRDye800CW as a Necrosis Avid Contrast Agent in Solid Tumors

Necrosis only occurs in pathological situations and is directly related to disease severity and, therefore, is an important biomarker. Tumor necrosis occurs in most solid tumors due to improperly functioning blood vessels that cannot keep up with the rapid growth, especially in aggressively growing tumors. The amount of necrosis per tumor volume is often correlated to rapid tumor proliferation and can be used as a diagnostic tool. Furthermore, efficient therapy against solid tumors will directly or indirectly lead to necrotic tumor cells, and detection of increased tumor necrosis can be an early marker for therapy efficacy. We propose the application of necrosis avid contrast agents to detect therapy-induced tumor necrosis. Herein, we advance gallium-68-labeled IRDye800CW, a near-infrared fluorescent dye that exhibits excellent necrosis avidity, as a potential PET tracer for in vivo imaging of tumor necrosis. We developed a reliable labeling procedure to prepare [68Ga]Ga-DOTA-PEG4-IRDye800CW ([68Ga]Ga-1) with a radiochemical purity of >96% (radio-HPLC). The prominent dead cell binding of fluorescence and radioactivity from [68Ga]Ga-1 was confirmed with dead and alive cultured 4T1-Luc2 cells. [68Ga]Ga-1 was injected in 4T1-Luc2 tumor-bearing mice, and specific fluorescence and PET signal were observed in the spontaneously developing tumor necrosis. The ip injection of D-luciferin enabled simultaneous bioluminescence imaging of the viable tumor regions. Tumor necrosis binding was confirmed ex vivo by colocalization of fluorescence uptake with TUNEL dead cell staining and radioactivity uptake in dichotomized tumors and frozen tumor sections. Our presented study shows that [68Ga]Ga-1 is a promising PET tracer for the detection of tumor necrosis.

Accuracy and safety of partial thickness femtosecond laser radial and arcuate keratotomy incisions in porcine eyes

Background To evaluate the accuracy and safety of micro radial and arcuate keratotomy incisions constructed by a femtosecond laser system with a curved contact patient interface in porcine eyes. Methods Partial thickness micro radial and arcuate keratotomy incisions were constructed in porcine eyes with a femtosecond laser system and evaluated for precision of depth, quality, and consistency. Optical coherence tomography was used to determine the accuracy and precision of incision depth. Corneal endothelial safety was assessed by a fluorescent live/dead cell viability assay to demonstrate laser-induced endothelial cell loss. Quality was evaluated by ease of opening and examination of interfaces. Results In two micro radial incision groups, intended incision depths of 50% and 80% resulted in mean achieved depths of 50.01% and 77.69%, respectively. In three arcuate incision groups, intended incision depths of 80%, 600 μm or 100 μm residual uncut bed thickness resulted in mean achieved depths of 80.16%, 603.03 μm and residual bed of 115 μm, respectively. No loss of endothelial cell density occurred when the residual corneal bed was maintained at a minimum of 85–116 µm. The incisions were easy to open, and interfaces were smooth. Conclusions A femtosecond laser system with curved contact interface created precise and reproducible micro radial and arcuate keratotomy incisions. Accuracy and precision of the incision depth and preservation of endothelial cell density demonstrated the effectiveness and safety of the system.

Bayesian calibration of a stochastic, multiscale agent-based model for predicting in vitro tumor growth

Hybrid multiscale agent-based models (ABMs) are unique in their ability to simulate individual cell interactions and microenvironmental dynamics. Unfortunately, the high computational cost of modeling individual cells, the inherent stochasticity of cell dynamics, and numerous model parameters are fundamental limitations of applying such models to predict tumor dynamics. To overcome these challenges, we have developed a coarse-grained two-scale ABM (cgABM) with a reduced parameter space that allows for an accurate and efficient calibration using a set of time-resolved microscopy measurements of cancer cells grown with different initial conditions. The multiscale model consists of a reaction-diffusion type model capturing the spatio-temporal evolution of glucose and growth factors in the tumor microenvironment (at tissue scale), coupled with a lattice-free ABM to simulate individual cell dynamics (at cellular scale). The experimental data consists of BT474 human breast carcinoma cells initialized with different glucose concentrations and tumor cell confluences. The confluence of live and dead cells was measured every three hours over four days. Given this model, we perform a time-dependent global sensitivity analysis to identify the relative importance of the model parameters. The subsequent cgABM is calibrated within a Bayesian framework to the experimental data to estimate model parameters, which are then used to predict the temporal evolution of the living and dead cell populations. To this end, a moment-based Bayesian inference is proposed to account for the stochasticity of the cgABM while quantifying uncertainties due to limited temporal observational data. The cgABM reduces the computational time of ABM simulations by 93% to 97% while staying within a 3% difference in prediction compared to ABM. Additionally, the cgABM can reliably predict the temporal evolution of breast cancer cells observed by the microscopy data with an average error and standard deviation for live and dead cells being 7.61±2.01 and 5.78±1.13, respectively.

Salt stress improves thermotolerance and high-temperature bioethanol production of multi-stress-tolerant Pichia kudriavzevii by stimulating intracellular metabolism and inhibiting oxidative damage

Background High-temperature bioethanol production benefits from yeast thermotolerance. Salt stress could induce obvious cross-protection against heat stress of Pichia kudriavzevii, contributing to the improvement of its thermotolerance and bioethanol fermentation. However, the underlying mechanisms of the cross-protection remain poorly understood. Results Salt stress showed obvious cross-protection for thermotolerance and high-temperature ethanol production of P. kudriavzevii observed by biomass, cell morphology and bioethanol production capacity. The biomass and ethanol production of P. kudriavzevii at 45 °C were, respectively, improved by 2.6 and 3.9 times by 300 mmol/L NaCl. Metabolic network map showed that salt stress obviously improved the key enzymes and intermediates in carbohydrate metabolism, contributing to the synthesis of bioethanol, ATP, amino acids, nucleotides, and unsaturated fatty acids, as well as subsequent intracellular metabolisms. The increasing trehalose, glycerol, HSPs, and ergosterol helped maintain the normal function of cell components. Heat stress induced serious oxidative stress that the ROS-positive cell rate and dead cell rate, respectively, rose from 0.5% and 2.4% to 28.2% and 69.2%, with the incubation temperature increasing from 30 to 45 °C. The heat-induced ROS outburst, oxidative damage, and cell death were obviously inhibited by salt stress, especially the dead cell rate which fell to only 20.3% at 300 mmol/L NaCl. The inhibiting oxidative damage mainly resulted from the abundant synthesis of GSH and GST, which, respectively, increased by 4.8 and 76.1 times after addition of 300 mmol/L NaCl. The improved bioethanol production was not only due to the improved thermotolerance, but resulted from the up-regulated alcohol dehydrogenases and down-regulated aldehyde dehydrogenases by salt stress. Conclusion The results provide a first insight into the mechanisms of the improved thermotolerance and high-temperature bioethanol production of P. kudriavzevii by salt stress, and provide important information to construct genetic engineering yeasts for high-temperature bioethanol production. Graphical Abstract

Development of a poly(arylene sulfide sulfone) antibacterial electrospun film as a skin wound dressing application

Tissue engineering has become a hot issue for skin wound healing because it can be used as an alternative treatment to traditional grafts. Nanofibrous films have been widely used due to their excellent properties. In this work, an organic/inorganic composite poly(arylene sulfide sulfone)/ZnO/graphene oxide (PASS/ZnO/GO) nanofibrous film was fabricated with the ZnO nanoparticles blending in an electrospun solution and post-treated with the GO deposition. The optimal PASS/ZnO/GO nanofibrous film was prepared by 2% ZnO nanoparticles, 3.0[Formula: see text]g/mL PASS electrospun solution, and 1% GO dispersion solution. The morphology, hydrophilicity, mechanical property, and cytotoxicity of the PASS/ZnO/GO nanofibrous film were characterized by using scanning electron microscopy, transmission electron microscope, water contact angle, tensile testing, and a Live/Dead cell staining kit. It is founded that the PASS/ZnO/GO nanofibrous film has outstanding mechanical properties and no cytotoxicity. Furthermore, the PASS/ZnO/GO nanofibrous film exhibits excellent antibacterial activity to both Escherichia coli and Staphylococcus aureus. Above all, this high mechanical property in the non-toxic and antibacterial nanofibrous film will have excellent application prospects in skin wound dressing.

Macrophage network dynamics depend on haptokinesis for optimal local surveillance

Macrophages are key immune cells with important roles for tissue surveillance in almost all mammalian organs. Cellular networks made up of many individual macrophages allow for optimal removal of dead cell material and pathogens in tissues. However, the critical determinants that underlie these population responses have not been systematically studied. Here, we investigated how cell shape and the motility of individual cells influences macrophage network responses in 3D culture settings and in mouse tissues. We show that surveying macrophage populations can tolerate lowered actomyosin contractility, but cannot easily compensate for a lack of integrin-mediated adhesion. Although integrins were dispensable for macrophage chemotactic responses, they were crucial to control cell movement and protrusiveness for optimal surveillance by a macrophage population. Our study reveals that β1 integrins are important for maintaining macrophage shape and network sampling efficiency in mammalian tissues, and sets macrophage motility strategies apart from the integrin-independent 3D migration modes of many other immune cell subsets.