No abstract

Worldwide efforts are currently trying to produce effective risk assessment models for orally ingested nanoparticles. These tests should provide quantitative information on the bioaccessibility and bioavailability of products of biotransformation, such as dissolved ionic species and/or aggregates. In vitro dissolution tests might be useful for nanoparticle risk assessment, because of their potential to quantitatively monitor the changes of specific properties (e.g., dissolution, agglomeration, etc.), which are critical factors linked to bioaccessibility/bioavailability. Unfortunately, the technological advancement of such tools is currently hampered by the complexity and evolving nature of nanoparticle properties that are strongly influenced by the environment and are often difficult to trace in a standardized manner. Hence, the test's success depends on its ability to quantify such properties using standardized experimental conditions to mimic reality as closely as possible. Here we applied an in vitro dissolution test to quantify the dissolution of silver nanoparticles under dynamic conditions, which likely occur in human digestion, providing a clear description of the bioaccessible ionic species (free and matrix bound ions or soluble silver organic or inorganic complexes) occurring during the different digestion phases. We demonstrated the test feasibility using a multi-technique approach and following pre-standardized operational procedures to allow for a comprehensive description of the process as a whole. Moreover, this can favour data reliability for benchmarking. Finally, we showed how the estimated values of the bioaccessible ionic species relate to absorption and excretion parameters, as measured in vivo. The outcomes presented in this work highlight the potential regulatory role of the dissolution test for orally ingested nanoparticles and, although preliminary, experimentally demonstrate the regulatory oriented "read-across" principle.

Albumin is one of the most abundant plasma proteins and is heavily glycated in diabetes. In this study, we have addressed whether variation in the albumin levels influence glycation of plasma proteins and HbA1c. The study was performed in three systems: (1) streptozotocin (STZ)-induced diabetic mice plasma, (2) diabetic clinical plasma, and (3) in vitro glycated plasma. Diabetic mice and clinical plasma samples were categorized as diabetic high albumin plasma (DHAP) and diabetic low albumin plasma (DLAP) on the basis of their albumin levels. For the in vitro experiment, two albumin levels, high albumin plasma (HAP) and low albumin plasma (LAP), were created by differential depletion of plasma albumin. Protein glycation was studied by using a combination of two-dimensional electrophoresis (2DE), Western blotting, and LC-MS(E). In both mice and clinical experiments, an increased plasma protein glycation was observed in DLAP than in DHAP. Additionally, plasma albumin levels were negatively correlated with HbA1c. The in vitro experiment with differential depletion of albumin mechanistically showed that the low albumin levels are associated with increased plasma protein glycation and that albumin competes for glycation with other plasma proteins.

A B S T R A C TInterferon-induced transmembrane proteins IFITM1 and IFITM3 (IFITM1/3) play a role in both RNA viral restriction and in human cancer progression. Using immunohistochemical staining of FFPE tissue, we identified subgroups of cervical cancer patients where IFITM1/3 protein expression is inversely related to metastasis. Guide RNA-CAS9 methods were used to develop an isogenic IFITM1/IFITM3 double null cervical cancer model in order to define dominant pathways triggered by presence or absence of IFITM1/3 signalling. A pulse SILAC methodology identified IRF1, HLA-B, and ISG15 as the most dominating IFNγ inducible proteins whose synthesis was attenuated in the IFITM1/IFITM3 double-null cells. Conversely, SWATH-IP mass spectrometry of ectopically expressed SBP-tagged IFITM1 identified ISG15 and HLA-B as dominant co-associated proteins. ISG15ylation was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. Proximity ligation assays indicated that HLA-B can interact with IFITM1/3 proteins in parental SiHa cells. Cell surface expression of HLA-B was attenuated in IFNγ treated IFITM1/IFITM3 double-null cells. SWATH-MS proteomic screens in cells treated with IFITM1-targeted siRNA cells resulted in the attenuation of an interferon regulated protein subpopulation including MHC Class I molecules as well as IFITM3, STAT1, B2M, and ISG15. These data have implications for the function of IFITM1/3 in mediating IFNγ stimulated protein synthesis including ISG15ylation and MHC Class I production in cancer cells. The data together suggest that pro-metastatic growth associated with IFITM1/3 negative cervical cancers relates to attenuated expression of MHC Class I molecules that would support tumor immune escape.Abbreviations: B2M, beta-2-microglobulin; FASP, filter-aided sample preparation; FA, formic acid; HLA, human leucocyte antigen; IFN, interferon; IFITM1/3, interferon-induced transmembrane receptors 1 and 3; ISG15, interferon-stimulated gene 15; IRF1, interferon regulatory factor 1; MHC, major histocompatibility complex; MS, mass spectrometry; NMWCO, nominal molecular weight cut-off; PBS, phosphate-buffered saline; PLA, proximity ligation assay; RT, room temperature; SWATH-IP, SWATH immunoprecipitation; SBP, twin streptavidin binding protein; UPLC, ultra performance liquid chromatography ⁎ Corresponding authors at