An integrative method for evaluating the biological effects of nanoparticle-protein corona.

BACKGROUND: Nanoplastics in the environment can enter the human body through gastrointestinal intake, dermal contact, and pulmonary inhalation, posing a threat to human health. Protein molecules in body fluids will quickly adsorb on the surfaces of the nanoplastics, forming a protein corona, which has implications for the interaction of the nanoplastics with cells and the metabolic pathways of the nanoplastic within cells. For years, practical tools such as dynamic light scattering, transmission electron microscopy, and liquid chromatography have been developed to understand the protein corona of nanoparticles (NPs), either in vitro or in cellular or molecular level. However, an integrated approach to understand the nanoparticles-protein corona is still lacking. METHODS: Using the most frequently observed environmental nanoplastics, polystyrene nanoplastics (PS), as a standard, we established an integrative structural characterization platform, a biophysical and biochemical evaluation method to investigate the effect of surface charge on protein corona composition. The cellular and molecular mechanisms were also explored through in vitro cellular experiments. RESULTS: The first integrative method for characterizing biological properties of NPs-protein corona has been established. This method comprehensively covers the critical aspects to understand NPs-protein corona interactions, from structure to function. CONCLUSIONS: The integrative method for nanoplastics microstructure characterization can be applied to the structural characterization of nanoparticles in nanoscale, which is of universal significance from in vitro characterization to cellular experiments and then to molecular mechanism studies. GENERAL SIGNIFICANCE: This strategy has high reliability and repeatability and can be applied both in environment and nanomedicine safety assessment.

Mitochondrial Voltage-Dependent Anion Channel 1-Hexokinase-II Complex-Targeted Strategy for Melanoma Inhibition Using Designed Multiblock Peptide Amphiphiles.

Targeted therapies of melanoma are of urgent need considering the resistance of this aggressive type of cancer to chemotherapeutics. The voltage-dependent anion channel 1 (VDAC1)-hexokinase-II (HK-II) complex is an emerging target for novel anticancer therapies based on induced mitochondria-mediated apoptosis. The low cell membrane permeability of the anticancer 12-mer peptide N-Ter (RDVFTKGYGFGL) derived from the N-terminal fragment of the VDAC1 protein impedes the intracellular targeting. Here, novel multiblock VDAC1-derived cationic amphiphilic peptides (referred to as Pal-N-Ter-TAT, pFL-N-Ter-TAT, and Pal-pFL-N-Ter-TAT) are designed with a self-assembly propensity and cell-penetrating properties. The created multiblock amphiphilic peptides of partial α-helical conformations form nanoparticles of ellipsoid-like shapes and are characterized by enhanced cellular uptake. The amphiphilic peptides can target mitochondria and dissociate the VDAC1-HK-II complex at the outer mitochondrial membrane, which result in mitochondria-mediated apoptosis. The latter is associated with decrease of the mitochondrial membrane potential, cytochrome c release, and changes of the expression levels of the apoptotic proteins in A375 melanoma cells. Importantly, the mitochondrial VDAC1-derived amphiphilic peptides have a comparable IC50 value for melanoma cells to a small-molecule drug, sorafenib, which has been previously used in clinical trials for melanoma. These results demonstrate the potential of the designed peptide constructs for efficient melanoma inhibition.

Seroepidemiology of Enterovirus 71 infection prior to the 2011 season in children in Shanghai.

BACKGROUND: In 2010, China experienced the largest outbreak on record of Enterovirus 71 (EV71)-associated Hand Foot and Mouth Disease (HFMD) with more than 1.7 million cases, 27,000 patients with severe neurological complications and 905 deaths. Understanding of the seroprevalence of neutralizing antibodies (NAb) against EV71 and their protective role against HFMD in children is crucial for the implementation of future therapeutic and prophylactic intervention. OBJECTIVES: To correlate the prevalence of NAb against EV71 genotype C4a in children prior to the 2011 epidemic season with severe EV71-associated HFMD disease during the subsequent 2011 epidemic season. STUDY DESIGN: 614 sera samples were collected from children without HFMD. EV71 NAb were tested by a quantitative PCR assay. Samples with NAb ≥1:8 were scored as positive. RESULTS: 122 (19.9%) of 614 sera were EV71-seropositive. The NAb seroprevalence was highest in infants 0-5 months of age (28.6%) and lowest in children 1-1.9 years of age (13.4%). 64.1% of severe EV71-associated HFMD occurred in children 1-2.9 years. CONCLUSIONS: Despite the large 2010 outbreak, the overall seroprevalence of EV71 in children is relatively low. The seropositive rate of EV71 NAb prior to the 2011 season was inversely correlated with the number of EV71-infected severe cases in 2011. Loss of maternal antibodies in infants and lack of acquired anti-EV71 immunity are responsible for increased proportion of severe HFMD in the 1-2 years age group. Our data suggest that future vaccination campaigns should be initiated as early as 6 months.

A new approach to monitor expression of aldo-keto reductase proteins in mouse tissues.

The aldo-keto reductase (AKR) proteins catalyze reduction of diverse aldehydes and play detoxification roles in many organisms. Since many substrates are shared among AKR, it is generally accepted that these enzymes can functionally compensate each other in response to oxidative stress. Their overall abundances are the important factor that partially reflects the capacity of antioxidant and detoxification in tissues. In this study, the strategy was proposed for generation of Pan-AKR antibodies to recognize most AKR proteins in mouse tissues. Derived from bioinformatic analysis, several consensus peptides with different potential antigenicities were synthesized, conjugated to hemocyanin from keyhole limpets and further delivered to rabbits to generate polyclonal antibodies. Three Pan-AKR antibodies exhibited the immune specificities and immune sensitivities, Pan-AKR-P1 for AKR1B and AKR1C, Pan-AKR-P3 for AKR1C and Pan-AKR-P4 for all the AKR proteins. Pan-AKR-P4 antibody was employed to 2-DE Western blot to examine the AKR abundances in mouse liver and kidney, resulting in seven immune-reactive spots from each tissue. Protein identification with MS revealed that most immune-positive spots were the members of AKR superfamily. Furthermore, Pan-AKR-P4 antibody was implemented to compare the different abundances of the AKR proteins in liver and kidney between normal and diabetic mice, suggesting that diabetes did cause some abnormal changes in the AKR protein abundances.

Systematic characterization of a novel gal operon in Thermoanaerobacter tengcongensis.

On the basis of the Thermoanaerobacter tengcongensis genome, a novel type of gal operon was deduced. The gene expression and biochemical properties of this operon were further characterized. RT-PCR analysis of the intergenic regions suggested that the transcription of the gal operon was continuous. With gene cloning and enzyme activity assays, TTE1929, TTE1928 and TTE1927 were identified to be GalT, GalK and GalE, respectively. Results elicited from polarimetry assays revealed that TTE1925, a hypothetical protein, was a novel mutarotase, termed MR-Tt. TTE1926 was identified as a regulator that could bind to two operators in the operon promoter. The transcriptional start sites were mapped, and this suggested that there are two promoters in this operon. Expression of the gal genes was significantly induced by galactose, whereas only MR-Tt expression was detected in glucose-cultured T. tengcongensis at both the mRNA and the protein level. In addition, the abundance of gal proteins was examined at different temperatures. At temperatures ranging from 60 to 80 degrees C, the level of MR-Tt protein was relatively stable, but that of the other gal proteins was dramatically decreased. The operator-binding complexes were isolated and identified by electrophoretic mobility shift assay-liquid chromatography (EMSA-LC) MS-MS, which suggested that several regulatory proteins, such as GalR and a sensory histidine kinase, participate in the regulation of the gal operon.

The responses of mitochondrial proteome in rat liver to the consumption of moderate ethanol: the possible roles of aldo-keto reductases.

A large body of evidence supports the view that mitochondria are a primary target of alcohol stress. Changes in mitochondrial proteins due to moderate ethanol intake, however, have not been broadly and accurately estimated. For this study, rats were fed low doses of ethanol and the mitochondria were isolated from heart, kidney, and liver, using ultracentrifugation with Nycodenz density gradient. The mitochondrial proteins were well resolved upon two-dimensional electrophoresis (2DE), and the alcohol-responsive 2DE spots were identified by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/TOF MS). Compared with the control group, the proteins extracted from liver mitochondria of ethanol-fed rats exhibited the significant changes on 2DE images, whereas the 2DE images obtained from the kidney and the heart mitochondria remained almost unchanged by ethanol feeding. Significantly, over 50% of the alcohol-responsive proteins in liver mitochondria were members of aldo-keto reductase family (AKR), which were usually present in cytoplasm. The organelle distributions of AKR proteins in liver mitochondria were further confirmed by Western blot analysis as well as by confocal microscopy. In addition, translocations of AKR were examined in the CHANG cell line, which was cultured with and without ethanol. The results of Western blot strongly suggested that the abundances of AKR proteins in the mitochondria were greatly reduced by the presence of ethanol in culture medium. The results of this study show that, even with moderate ethanol feeding, the mitochondrial proteome in rat liver was more sensitive to alcohol stress than that of either the kidney or the heart. The translocation of AKR proteins may be involved in the detoxification of liver cells.