Development and validation of a flexible DNA extraction (PAN) method for liquid biopsy of multiple sample types.

BACKGROUND: Liquid biopsy is gaining increasing popularity in cancer screening and diagnosis. However, there is no relatively mature DNA isolation method or commercial kit available that is compatible with different LB sample types. This study developed a PAN-sample DNA isolation method (PAN method) for liquid biopsy samples. METHODS: The PAN method has two key steps, including biosample-specific pretreatments for various LB sample types and high concentration guanidine thiocyanate buffer for lysis and denaturation procedure. Subsequently, the performance of PAN method was validated by a series of molecular analyses. RESULTS: The PAN method was used to isolate DNA from multiple sample types related to LB, including plasma, serum, saliva, nasopharyngeal swab, and stool. All purified DNA products showed good quality and high quantity. Comparison of KRAS mutation analysis using DNA purified using PAN method versus QIAamp methods showed similar efficiency. Epstein-Barr virus DNA was detected via Q-PCR using DNA purified from serum, plasma, nasopharyngeal swab, and saliva samples collected from nasopharyngeal carcinoma patients. Similarly, methylation sequencing of swab and saliva samples revealed good coverage of target region and high methylation of HLA-DPB1 gene. Finally, 16S rDNA gene sequencing of saliva, swab, and stool samples successfully defines the relative abundance of microbial communities. CONCLUSIONS: This study developed and validated a PAN-sample DNA isolation method that can be used for different LB samples, which can be applied to molecular epidemiological research and other areas.

Environmentally persistent free radicals on photoaged microplastics from disposable plastic cups induce the oxidative stress-associated toxicity.

Microplastics (MPs) are ubiquitous environmental contaminants that exerting multiple toxicological effects. Most studies have focused primarily on the models of unaged MPs and lack environmental relevance. The generation and toxicity of environmentally persistent free radicals (EPFRs) on photoaging MPs from disposable plastic cups (DPC-MPs) have not been well studied. Here, the formation of EPFRs on photoaged DPC-MPs and their toxic effects in nematodes were investigated. UV irradiation generated EPFRs, which influenced the characterization of DPC-MPs. Exposure to photoaged DPC-MPs at environmentally relevant concentrations （100-1000 µg/L） reduced the locomotion behavior, body length, and brood size. The Reactive oxygen species (ROS) production, lipofuscin accumulation, malondialdehyde (MDA), and 8-hydroxy-2'-deoxyguanosine (8-OHdG) levels were increased along with the downregulation of the expression levels of associated genes, such as clk-1, clt-1, and gst-4，in nematodes. Moreover, the toxicity and oxidative stress response of nematodes were significantly inhibited due to N-acetyl-l-cysteine (NAC). Pearson's correlation analysis revealed that the oxidative stress was significantly associated with adverse physiological effects. Therefore, EPFRs on photoaged DPC-MPs cause toxicity in nematodes, and oxidative stress is important for regulating toxicity. This study offers novel insights into the potential risks of DPC-MPs under UV irradiation, highlighting the need to consider the role of EPFRs in toxicity assessments of DPC-MPs.

Photoaging enhances combined toxicity of microplastics and tetrabromobisphenol A by inducing intestinal damage and oxidative stress in Caenorhabditis elegans.

Microplastics (MPs) are emerging environmental contaminants that often co-exist with tetrabromobisphenol A (TBBPA) in the environment. However, the joint effect of TBBPA and photoaged MPs at ambient concentrations remains unknown largely. In this study, the combined toxicity of ultraviolet-aged polystyrene (UV-PS) and TBBPA was investigated in Caenorhabditis elegans. UV irradiation could change the physical and chemical characteristics of polystyrene (PS), and UV-PS (90.218 µg/g) showed a stronger adsorption capacity than PS of 79.424 µg/g. Toxicity testing showed that 1 µg/L UV-PS enhanced the toxic effect of 1 µg/L TBBPA by reducing body length, locomotion behavior, and brood size in nematodes. Using ROS production, lipofuscin accumulation, and expression of gst-4::GFP as endpoints, the combined exposure of UV-PS and TBBPA induced stronger oxidative stress than TBBPA alone. Joint exposure to UV-PS and TBBPA significantly increased of Nile red and blue food dye in its intestinal tract compared to that in the TBBPA exposure group, indicating that co-exposure enhanced intestinal permeability. After co-exposure to UV-PS and TBBPA, the expression of the associated genes detected increased significantly. Therefore, UV-PS enhances the adverse effects of TBBPA through intestinal damage and oxidative stress in nematodes. These findings suggest that the co-presence of photoaged PS and TBBPA results in high environmental risks.

Generation of environmentally persistent free radicals on photoaged tire wear particles and their neurotoxic effects on neurotransmission in Caenorhabditis elegans.

Tire wear particles (TWP) are a prevalent form of microplastics (MPs) extensively distributed in the environment, raising concerns about their environmental behaviors and risks. However, knowledge regarding the properties and toxicity of these particles at environmentally relevant concentrations, specifically regarding the role of environmentally persistent free radicals (EPFRs) generated during TWP photoaging, remains limited. In this study, the evolution of EPFRs on TWP under different photoaging times and their adverse effects on Caenorhabditis elegans were systematically investigated. The photoaging process primarily resulted in the formation of EPFRs and reactive oxygen species (O2•-, ⋅OH, and 1O2), altering the physicochemical properties of TWP. The exposure of nematodes to 100 µg/L of TWP-50 (TWP with a photoaging time of 50 d) led to a significant decrease in locomotory behaviors (e.g., head thrashes, body bends, and wavelength) and neurotransmitter contents (e.g., dopamine, glutamate, and serotonin). Similarly, the expression of neurotransmission-related genes was reduced in nematodes exposed to TWP-50. Furthermore, the addition of free-radical inhibitors significantly suppressed TWP-induced neurotoxicity. Notably, correlation analysis revealed a significantly negative correlation between EPFRs levels and the locomotory behaviors and neurotransmitter contents of nematodes. Thus, it was concluded that EPFRs on photoaged TWP induce neurotoxicity by affecting neurotransmission. These findings elucidate the toxicity effects and mechanisms of EPFRs, emphasizing the importance of considering their contributions when evaluating the environmental risks associated with TWP.

Amino carboxymethyl chitosan//dialdehyde starch/polyvinyl alcohol double-layer film loaded with ε-polylysine.

To develop food packaging with good antibacterial activity and mechanical performance, four amino carboxymethyl chitosan (ACC)//dialdehyde starch (DAS) /polyvinyl alcohol (PVA) films were prepared by Schiff base and hydrogen bond interactions for efficient loading and release of ε-polylysine (ε-PL). The effects of the Schiff base reaction on the physicochemical properties of the films were explored based on the different aldehyde group contents in DAS. The ACC//DAS4/PVA film exhibited a tensile strength of 62.5 MPa, and the water vapor and oxygen permeability was 8.77 × 10-3·g·mm/m2·d·kPa and 0.15 × 103·cm3·mm/m2·d, respectively. By leveraging the Schiff base reaction, the film swelling properties were improved by adjusting the cross-link density, mesh size, and molecular mass between the cross-links. The ACC//DAS4/PVA film could efficiently load ε-PL with a value of 98.44% and long-term release in a food simulant of 10% ethanol at 25 °C for 120 min. Moreover, the ACC-ε-PL//DAS4/PVA film was successfully used for salmon preservation.

The generation of environmentally persistent free radicals on photoaged microbeads from cosmetics enhances the toxicity via oxidative stress.

Microbeads used in personal care products have been one of the important sources of microplastics (MPs), and little has been reported on their environmental behaviors and health risks. The characteristics of environmentally persistent free radicals (EPFRs) and the toxicity assessment of MPs (environmentally relevant concentrations) from cosmetics during photoaging remains largely unknown. In this study, the formation of EPFRs on polyethylene (PE) microbeads from facial scrubs under light irradiation and their toxicity were investigated using C. elegans as a model organism. The results suggested that light irradiation induced the generation of EPFRs, which accelerates the aging process and alters the physicochemical properties of PE microbeads. Acute exposure to PE (1 mg/L) at photoaged times of 45-60 d significantly decreased the physiological indicators (e.g., head thrashes, body bends, and brood size). The oxidative stress response and stress-related gene expression were also enhanced in nematodes. The addition of N-acetyl-l-cysteine induced significant inhibition of toxicity and oxidative stress in nematodes exposed to 45-60 d of photoaged PE. The Pearson correlation results showed that the concentration of EPFRs was significantly correlated with physiological indicators, oxidative stress, and related-genes expression in nematodes. The data confirmed that the generation of EPFRs combined with heavy metals and organics contributed to toxicity induced by photoaged PE, and oxidative stress might be involved in regulating adverse effects in C. elegans. The study provides new insight into the potential risks of microbeads released into the environment during photoaging. The findings also highlight the necessity for considering the role of EPFRs formation in evaluating the impacts of microbeads.

Preparation of chitosan-cellulose-benzyl isothiocyanate nanocomposite film for food packaging applications.

To improve the mechanical and barrier properties of cellulose and chitosan (CS) and to allow the application of volatile antibacterial benzyl isothiocyanate (BITC) in active packaging, a double-layer nanocomposite film was prepared. Cellulose nanofibers (CNF) were crosslinked with CS via genipin to build the substrate. Quaternized cellulose nanocrystals (QCNC) were synthesized for carrying BITC as the coating material. By the layer-by-layer self-assembly approach, CS-CNF/QCNC-BITC film was fabricated. This film possessed the tensile strength of 33.75 MPa, low permeabilities of oxygen (6.9 × 10-17 m3/s·m·Pa) and moisture (1.2 × 10-11 g/s·m·Pa), and good antibacterial activity with the inhibition zone diameters of 4.9, 4.2 and 2.7 cm against Escherichia coli, Salmonella typhimurium and Staphylococcus aureus. The total viable count, total volatile basic nitrogen and thiobarbituric acid-reactive substances of the chicken wrapped CS-CNF/QCNC-BITC were only 4.4 log CFU/g, 17.7 mg/100 g and 0.44 mg/kg at 14 days, indicating a potential application of CS-CNF/QCNC-BITC for food packaging.

Flocculation of polymer stabilized nanocrystal suspensions to produce redispersible powders.

Aqueous suspensions of crystalline naproxen nanoparticles, formed by antisolvent precipitation, were flocculated with sodium sulfate, filtered, and dried to form redispersible powders for oral delivery. The particles were stabilized with polyvinylpyrrolidone (PVP K-15) and/or poly(ethylene oxide-b-propylene oxide-b-ethylene oxide) (poloxamer 407). The yield of the drug in the powder was typically 92-99%, and the drug loading was reproducible to within 1-2%. The filtration process increased the drug loading by up to 61% relative to the initial value, as unbound surfactant was removed with the filtrate. Upon redispersion of the dried powder, the average particle size measured by light scattering was comparable to the original value in the aqueous suspension prior to flocculation, and consistent with primary particle sizes observed by scanning electron microscopy (SEM). For 300-nm particles, up to 95% of the drug dissolved in 2 min. The dissolution rate was correlated linearly with the specific surface area calculated from the average particle diameter after redispersion. The redispersion of dried powders was examined as a function of the salt concentration used for flocculation and the surfactant composition and concentration. Flocculation followed by filtration and drying is an efficient and highly reproducible process for the rapid recovery of drug nanoparticles to produce wettable powders with high drug loading and rapid dissolution.

Triplex DNA Nanoswitch for pH-Sensitive Release of Multiple Cancer Drugs.

A DNA-based stimulus-responsive drug delivery system for synergetic cancer therapy has been developed. The system is built on a triplex-DNA nanoswitch capable of precisely responding to pH variations in the range of ∼5.0-7.0. In extracellular neutral pH space, the DNA nanoswitch keeps a linear conformation, immobilizing multiple therapeutics such as small molecules and antisense compounds simultaneously. Following targeted cancer cell uptake via endocytosis, the nanoswitch inside acidic intracellular compartments goes through a conformational change from linear to triplex, leading to smart release of the therapeutic combination. This stimuli-responsive drug delivery system does not rely on artificial responsive materials, making it biocompatible. Furthermore, it enables simultaneous delivery of multiple therapeutics for enhanced efficacy. Using tumor-bearing mouse models, we show efficient gene silencing and significant inhibition of tumor growth upon intravenous administration of the smart nanoswitch, providing opportunities for combinatorial cancer therapy.

Enhanced electron transfer for hemoglobin entrapped in a cationic gemini surfactant films on electrode and the fabrication of nitric oxide biosensor.

The direct electrical communication between hemoglobin (Hb) and GCE surface was achieved based on the immobilization of Hb in a cationic gemini surfactant film and characterized by electrochemical techniques. The cyclic voltammograms showed that direct electron transfer between Hb and electrode surface was obviously promoted and then a novel unmediated nitric oxide (NO) biosensor was constructed in view of this protein-based electrode. This modified electrode showed an enzyme-like activity towards the reduction of NO and its amperometric response to NO was well-behaved with a rapid response time and displaying Michaelis-Menten kinetics with a calculated Km(app) value of 84.37 micromol L(-1). The detection limit was estimated to be 2.00 x 10(-8)mol L(-1). This biosensor was behaving as expected that it had a good stability and reproducibility, a higher sensitivity and selectivity and should has a potential application in monitoring NO released from biologic samples.

Preparation of cyclosporine A nanoparticles by evaporative precipitation into aqueous solution.

Amorphous nanoparticle suspensions of a poorly water-soluble drug, cyclosporine A, are produced by a new process, evaporative precipitation into aqueous solution (EPAS). The rapid evaporation of a heated organic solution of the drug, which is atomized into an aqueous solution, results in fast nucleation leading to nanoparticles suspensions. Hydrophilic stabilizers, introduced in the organic or aqueous phases, limit particle growth and inhibit crystallization for drug concentrations as high as 35 mg/ml, and drug/surfactant ratios up to 1.0. The suspensions may be used in parenteral formulations to enhance bioavailability or may be dried to produce oral dosage forms with the potential for high dissolution rates due to the low crystallinity, small particle size and hydrophilic stabilizer that enhances wetting.

TEMPO-oxidized cellulose hydrogel as a high-capacity and reusable heavy metal ion adsorbent.

Nitroxy radical catalyzed oxidation with hypochlorite/bromide (TEMPO-mediated oxidation) was performed on a cellulose hydrogel prepared using LiOH/urea solvent. TEMPO oxidation successfully introduced carboxyl groups onto the surface of the cellulose hydrogel with retention of the gel structure and its nanoporous property. The equilibrium measurement of Cu(2+) adsorption showed favorable interaction with Cu(2+) and high maximum adsorption capacity. In addition, over 98% of the adsorbed Cu(2+) was recovered using acid treatment, and the subsequent washing allowed the TEMPO-oxidized gels to be used repeatedly. Furthermore, the TEMPO-oxidized cellulose hydrogel showed high adsorption capacity for other toxic metal ions such as Zn(2+), Fe(3+), Cd(2+), and Cs(+).