Engineering Antimicrobial Metal-Phenolic Network Nanoparticles with High Biocompatibility for Wound Healing.

Antibiotic-resistant bacteria pose a global health threat by causing persistent and recurrent microbial infections. To address this issue, antimicrobial nanoparticles (NPs) with low drug resistance but potent bactericidal effects have been developed. However, many of the developed NPs display poor biosafety and their synthesis often involves complex procedures and the antimicrobial modes of action are unclear. Herein, a simple strategy is reported for designing antimicrobial metal-phenolic network (am-MPN) NPs through the one-step assembly of a seeding agent (diethyldithiocarbamate), natural polyphenols, and metal ions (e.g., Cu2+ ) in aqueous solution. The Cu2+ -based am-MPN NPs display lower Cu2+ antimicrobial concentrations (by 10-1000 times) lower than most reported nanomaterials and negligible toxicity across various models, including, cells, blood, zebrafish, and mice. Multiple antimicrobial modes of the NPs have been identified, including bacterial wall disruption, reactive oxygen species production, and quinoprotein formation, with the latter being a distinct pathway identified for the antimicrobial activity of the polyphenol-based am-MPN NPs. The NPs exhibit excellent performance against multidrug-resistant bacteria (e.g., methicillin-resistant Staphylococcus aureus (MRSA)), efficiently inhibit and destroy bacterial biofilms, and promote the healing of MRSA-infected skin wounds. This study provides insights on the antimicrobial properties of metal-phenolic materials and the rational design of antimicrobial metal-organic materials.

Combined Hepatotoxicity and Toxicity Mechanism of Intermedine and Lycopsamine.

Pyrrolizidine alkaloids (PAs) are common constituents of plants and have serious hepatotoxicity. Intermedine (Im) and lycopsamine (La) are two monoesters of PAs that frequently coexist in the PA-containing plants (e.g., comfrey and tea). The present study aimed to explore the combined hepatotoxicity and toxicity mechanism of the Im and La mixture. In vitro, the combined cytotoxicity of the Im and La mixture on human hepatocytes (HepD) was examined by CCK-8, colony formation, wound healing, and Annexin V/PI staining assays. The combination of Im and La inhibited the ability of HepD cells to proliferate, colonize, and migrate and induced hepatocytes apoptosis in a dose-dependent manner. In addition to significantly causing a burst of intracellular reactive oxygen species (ROS), mitochondrial apoptosis, and endoplasmic reticulum (ER) stress, the Im and La mixture can also cause an increase in intracellular Ca2+, triggering the PERK/eIF2α/ATF4/CHOP apoptosis pathway. This study provided the first direct evidence that the combined PAs induced hepatotoxicity through ER-mediated apoptosis. These results supplemented the basic toxicity data for the combined PAs and provided a new perspective for the risk assessment of combined PA toxicity.

3D printed PCLA scaffold with nano-hydroxyapatite coating doped green tea EGCG promotes bone growth and inhibits multidrug-resistant bacteria colonization.

OBJECTIVES: 3D-printing scaffold with specifically customized and biomimetic structures gained significant recent attention in tissue engineering for the regeneration of damaged bone tissues. However, constructed scaffolds that simultaneously promote bone regeneration and in situ inhibit bacterial proliferation remains a great challenge. This study aimed to design a bone repair scaffold with in situ antibacterial functions. MATERIALS AND METHODS: Herein, a general strategy is developed by using epigallocatechin-3-gallate (EGCG), a major green tea polyphenol, firmly anchored in the nano-hydroxyapatite (HA) and coating the 3D printed polymerization of caprolactone and lactide (PCLA) scaffold. Then, we evaluated the stability, mechanical properties, water absorption, biocompatibility, and in vitro antibacterial and osteocyte inductive ability of the scaffolds. RESULTS: The coated scaffold exhibit excellent activity in simultaneously stimulating osteogenic differentiation and in situ resisting methicillin-resistant Staphylococcus aureus colonization in a bone repair environment without antibiotics. Meanwhile, the prepared 3D scaffold has certain mechanical properties (39.3 ± 3.2 MPa), and the applied coating provides the scaffold with remarkable cell adhesion and osteogenic conductivity. CONCLUSION: This study demonstrates that EGCG self-assembled HA coating on PCLA surface could effectively enhance the scaffold's water absorption, osteogenic induction, and antibacterial properties in situ. It provides a new strategy to construct superior performance 3D printed scaffold to promote bone tissue regeneration and combat postoperative infection in situ.

Hepatotoxicity of Pyrrolizidine Alkaloid Compound Intermedine: Comparison with Other Pyrrolizidine Alkaloids and Its Toxicological Mechanism.

Pyrrolizidine alkaloids (PAs) are common secondary plant compounds with hepatotoxicity. The consumption of herbal medicines and herbal teas containing PAs is one of the main causes of hepatic sinusoidal obstruction syndrome (HSOS), a potentially life-threatening condition. The present study aimed to reveal the mechanism underlying the cytotoxicity of intermedine (Im), the main PA in Comfrey. We evaluated the toxicity of the retronecine-type PAs with different structures to cell lines derived from mammalian tissues, including primary mouse hepatocytes, human hepatocytes (HepD), mouse hepatoma-22 (H22) and human hepatocellular carcinoma (HepG2) cells. The cytotoxicity of Im to hepatocyte was evaluated by using cell counting kit-8 assay, colony formation experiment, wound healing assay and dead/live fluorescence imaging. In vitro characterization showed that these PAs were cytotoxic and induced cell apoptosis in a dose-dependent manner. We also demonstrated that Im induced cell apoptosis by generating excessive reactive oxygen species (ROS), changing the mitochondrial membrane potential and releasing cytochrome c (Cyt c) before activating the caspase-3 pathway. Importantly, we directly observed the destruction of the cell mitochondrial structure after Im treatment through transmission electron microscopy (TEM). This study provided the first direct evidence of Im inducing hepatotoxicity through mitochondria-mediated apoptosis. These results supplemented the basic toxicity data of PAs and facilitated the comprehensive and systematic evaluation of the toxicity caused by PA compounds.

Prognostic and diagnostic roles of prolyl 4-hydroxylase subunit α members in breast cancer.

Aim: We aimed to evaluate the diagnostic and prognostic values of P4HAs in breast cancer (BC) patients. Materials & methods: Kaplan-Meier plotter was used to evaluate the prognostic values of P4HAs and correlations between their expression and clinical characteristics were assessed based on The Cancer Genome Atlas and the Human Protein Atlas. Results: The current study showed that P4HAs were highly expressed in BC patients with clinical stage I compared with nontumor control and elevated P4HAs were correlated with poor survival outcomes. Subtypes analysis revealed that P4HA1 and P4HA2 were most expressed in HER2+ subtypes patients. Univariate analysis displayed that elevated P4HA1 and P4HA3 correlated with unfavorable recurrence-free survival in mutated TP53 patients. Conclusion: This study indicated the diagnostic and prognostic roles of P4HAs members and broadened the biomarker fields of early diagnosis and prognostic monitoring of BC patients.

Physiological, biochemical and transcription effects of roxithromycin before and after phototransformation in Chlorella pyrenoidosa.

Photodegradation is an important transformation pathway for macrolide antibiotics (MCLs) in aquatic environments, but the ecotoxicity of MCLs after phototransformation has not been reported in detail. This study investigated the effects of roxithromycin (ROX) before and after phototransformation on the growth and physio-biochemical characteristics of Chlorella pyrenoidosa, and its toxicity were explored using transcriptomics analysis. The results showed that 2 mg/L ROX before phototransformation (T0 group) inhibited algae growth with inhibition rates of 53.06%, 54.17%, 47.26%, 31.27%, and 28.38% at 3, 7, 10, 14, and 21 d, respectively, and chlorophyll synthesis was also inhibited. The upregulation of antioxidative enzyme activity levels and the malondialdehyde content indicated that ROX caused oxidative damage to C. pyrenoidosa during 21 d of exposure. After phototransformation for 48 h (T48 group), ROX exhibited no significant impact on the growth and physio-biochemical characteristics of the microalgae. Compared with the control group (without ROX and its phototransformation products), 2010 and 2988 differentially expressed genes were identified in the T0 and T48 treatment groups, respectively. ROX significantly downregulated genes related to porphyrin and chlorophyll metabolism, which resulted in the inhibition of chlorophyll synthesis and algae growth. ROX also significantly downregulated genes of DNA replication, suggesting the increased DNA proliferation risks in algae. After phototransformation, ROX upregulated most of the genes associated with the porphyrin and chlorophyll metabolism pathway, which may be the reason that the chlorophyll content in T48 treatment group showed no significant difference from the control group. Almost all light-harvesting chlorophyll a/b (LHCa/b) gene family members were upregulated in both T0 and T48 treatment groups, which may compensate part of the stress of ROX and its phototransformation products.

[Analysis of genetic variants in a pedigree affected with hereditary multiple osteochondroma].

OBJECTIVE: To explore the genetic basis for a pedigree affected with hereditary multiple osteochondroma (HMO). METHODS: Peripheral blood samples were collected from the proband and members of his pedigree with informed consent. Following extraction of genomic DNA, all coding exons and flanking intronic sequences (-10 bp) of the EXT1 and EXT2 genes were subjected to targeted capture and next generation sequencing (NGS). Suspected variant was verified by Sanger sequencing. RESULTS: A heterozygous nonsense variant (c.1911C>A) was found in exon 10 of the EXT1 gene in the proband and his affected father but not in a healthy sister and normal controls. The variant was classified as a pathogenic based on the guidelines of the American College of Medical Genetics and Genomics (PVS1+PM2+PP1). Bioinformatic analysis predicted that the c.1911C>A variant may be disease-causing via nonsense-mediated mRNA decay and anomalous splicing. CONCLUSION: The c.1911C>A variant probably underlay the disease in this pedigree. Discovery of this variant enriched the variant spectrum of HMO.

Qualitative and Quantitative Analysis of Tumor Cell Invasion Using Au Clusters.

Tumor invasion/metastasis is still the major cause of death in cancer patients. Membrane type-1 matrix metalloproteinase (MT1-MMP) is directly related to tumor invasion/metastasis. To accurately and quickly distinguish the risk of invasion/metastasis of primary tumor cells, it is urgent to develop a simple and precise quantitative method to distinguish the expression level of MT1-MMP. In this work, we have constructed red fluorescent Au clusters with peroxidase-like properties that could specifically bind to MT1-MMP on human cervical cancer cells. After MT1-MMP was labelled with Au clusters, we could visually see red fluorescence of MT1-MMP on cervical cancer cells via fluorescence microscopy and catalytic color imaging using an ordinary optical microscope. The constructed Au clusters contained 26 Au atoms; thus, the amount of MT1-MMP on cervical cancer cells could be accurately quantified using inductively coupled plasma mass spectrometry (ICP-MS). More importantly, the invasion/metastasis capabilities of the cervical cancer Siha, Caski and Hela cells with different MT1-MMP amounts could be accurately distinguished by fluorescence/catalysis qualitative imaging and ICP-MS quantitative analysis. This method of qualitative/quantitative analysis of tumor-associated proteins on cancer cells has great potential for accurately diagnosing aggressive tumor cells and assessment of their invasion/metastasis risk.

Rapid measurement of total polyphenol content in tea by kinetic matching approach on microfluidic paper-based analytical devices.

In this work, a facile kinetic matching approach for total polyphenol content (TPC) measurement was developed based on the adoption of microfluidic paper-based analytical devices with symmetric channel distribution. A set of Folin-Ciocalteu reactions performed on the same paper chip were activated all at the same time through synchronized filling of sodium carbonate solution among individual channels. Gallic acid was found valid as a standard compound for kinetic matching measurement of tea samples. TPC of tea infusions was successfully measured within ten minutes without any complexed time control procedure needed. Under the optimized conditions, the new developed method showed good linearity in the TPC range of 10-100 mg/L (r > 0.9955) and the inter-chip precision was 5.6% (n = 11). The results measured with the new developed approach were in good agreement with those with the conventional FC assay.

Identification of Potential Diagnostic and Prognostic Values of P4HA1 Expression in Lung Cancer, Breast Cancer, and Head and Neck Cancer.

The aims of this study were to investigate the expression of prolyl 4-hydroxylase subunit alpha-1 (P4HA1) and its relationship with clinicopathological features in lung cancer (LC), breast cancer (BC), and head and neck cancer (HNSC) and to discuss the possibility of P4HA1 being a potential diagnostic and prognostic biomarker. Data on the RNA expression profile, protein expression profile, and relevant clinical information were downloaded from The Cancer Genome Atlas (TCGA) and The Human Protein Atlas databases. The relationship between P4HA1 mRNA expression and clinicopathological features was evaluated. Survival analysis was performed to assess overall survival (OS) and relapse-free survival (RFS). The multivariate Cox regression model was employed to analyze the independent prognostic factors. Finally, protein-protein interaction networks were constructed and enrichment analysis was performed to identify the latent P4HA1-related terms and pathways. This study showed that P4HA1 was upregulated in three types of tumor tissues (p < 0.05) and high P4HA1 was significantly relevant to the clinical features of patients with LC, BC, or HNSC. Survival analysis indicated that patients with high P4HA1 had unfavorable clinical outcomes. Multivariate analysis showed that the high P4HA1 expression was an independent prognostic factor for poor OS and RFS in LC and HNSC patients. Bioinformatic analysis was performed to predict P4HA1-interacted proteins and further evaluate possible signal pathways. In the current study, the rising P4HA1 was identified in LC, BC, and HNSC and significantly correlated with the clinicopathological features of patients. High P4HA1, suggesting poor clinical outcomes, could be used as an early diagnostic and prognostic biomarker for patients with aforementioned tumors.

A facile microfluidic paper-based analytical device for acetylcholinesterase inhibition assay utilizing organic solvent extraction in rapid detection of pesticide residues in food.

The incompatibility of most organic solvents with acetylcholinesterase (AChE) inhibition assay normally limits pesticide extraction efficiency in sample pretreatment, which might cause false negatives in real world sample assessment. Herein, a novel method has been developed for an improved AChE inhibition assay via organic solvent extraction combined spontaneous in situ solvent evaporation on microfluidic paper-based analytical devices. Enzyme pre-immobilization procedure was spared and AChE was added to the system after sampling step until a complete in-situ solvent evaporation process was performed on chip. IC50 levels of the six investigated organophosphate and carbamate pesticides indicated a completely eliminated influence of solvents on AChE behavior with the new method. Most importantly, analytical performances were significantly improved in food sample measurements. Reduction in matrix effect was observed when acetonitrile was adopted for lettuce sample pretreatment instead of water. Studies on different pesticides suggested a remarkably decreased discrimination effect on recoveries from sample pretreatment with the new developed method. The recovery level for phoxim spiked head lettuce samples reached (107.5 ± 14.2) %, in comparison with that of (18.6 ± 1.4) % from water-based extraction. Spiked water and apple juice samples with carbaryl concentration of as low as 0.02 mg L-1 were also successfully recognized with the present method by visual detection. This is the first report on direct sampling of organic extracts for AChE inhibition assay on-chip and it might provide a new perspective for real world sample assessments involving bio-reagents.