Effect of Sample Transportation on the Proteome of Human Circulating Blood Extracellular Vesicles.

Circulating extracellular vesicles (cEV) are released by many kinds of cells and play an important role in cellular communication, signaling, inflammation modulation, coagulation, and tumor growth. cEV are of growing interest, not only as biomarkers, but also as potential treatment targets. However, very little is known about the effect of transporting biological samples from the clinical ward to the diagnostic laboratory, notably on the protein composition. Pneumatic tube systems (PTS) and human carriers (C) are both routinely used for transport, subjecting the samples to different ranges of mechanical forces. We therefore investigated qualitatively and quantitatively the effect of transport by C and PTS on the human cEV proteome and particle size distribution. We found that samples transported by PTS were subjected to intense, irregular, and multidirectional shocks, while those that were transported by C mostly underwent oscillations at a ground frequency of approximately 4 Hz. PTS resulted in the broadening of nanoparticle size distribution in platelet-free (PFP) but not in platelet-poor plasma (PPP). Cell-type specific cEV-associated protein abundances remained largely unaffected by the transport type. Since residual material of lymphocytes, monocytes, and platelets seemed to dominate cEV proteomes in PPP, it was concluded that PFP should be preferred for any further analyses. Differential expression showed that the impact of the transport method on cEV-associated protein composition was heterogeneous and likely donor-specific. Correlation analysis was nonetheless able to detect that vibration dose, shocks, and imparted energy were associated with different terms depending on the transport, namely in C with cytoskeleton-regulated cell organization activity, and in PTS with a release of extracellular vesicles, mainly from organelle origin, and specifically from mitochondrial structures. Feature selection algorithm identified proteins which, when considered together with the correlated protein-protein interaction network, could be viewed as surrogates of network clusters.

Inference of kinase-signaling networks in human myeloid cell line models by Phosphoproteomics using kinase activity enrichment analysis (KAEA).

BACKGROUND: Despite the introduction of targeted therapies, most patients with myeloid malignancies will not be cured and progress. Genomics is useful to elucidate the mutational landscape but remains limited in the prediction of therapeutic outcome and identification of targets for resistance. Dysregulation of phosphorylation-based signaling pathways is a hallmark of cancer, and therefore, kinase-inhibitors are playing an increasingly important role as targeted treatments. Untargeted phosphoproteomics analysis pipelines have been published but show limitations in inferring kinase-activities and identifying potential biomarkers of response and resistance. METHODS: We developed a phosphoproteomics workflow based on titanium dioxide phosphopeptide enrichment with subsequent analysis by liquid chromatography tandem mass spectrometry (LC-MS). We applied a novel Kinase-Activity Enrichment Analysis (KAEA) pipeline on differential phosphoproteomics profiles, which is based on the recently published SetRank enrichment algorithm  with reduced false positive rates. Kinase activities were inferred by this algorithm using an extensive reference database comprising five experimentally validated kinase-substrate meta-databases complemented with the NetworKIN in-silico prediction tool. For the proof of concept, we used human myeloid cell lines (K562, NB4, THP1, OCI-AML3, MOLM13 and MV4-11) with known oncogenic drivers and exposed them to clinically established kinase-inhibitors. RESULTS: Biologically meaningful over- and under-active kinases were identified by KAEA in the unperturbed human myeloid cell lines (K562, NB4, THP1, OCI-AML3 and MOLM13). To increase the inhibition signal of the driving oncogenic kinases, we exposed the K562 (BCR-ABL1) and MOLM13/MV4-11 (FLT3-ITD) cell lines to either Nilotinib or Midostaurin kinase inhibitors, respectively. We observed correct detection of expected direct (ABL, KIT, SRC) and indirect (MAPK) targets of Nilotinib in K562 as well as indirect (PRKC, MAPK, AKT, RPS6K) targets of Midostaurin in MOLM13/MV4-11, respectively. Moreover, our pipeline was able to characterize unexplored kinase-activities within the corresponding signaling networks. CONCLUSIONS: We developed and validated a novel KAEA pipeline for the analysis of differential phosphoproteomics MS profiling data. We provide translational researchers with an improved instrument to characterize the biological behavior of kinases in response or resistance to targeted treatment. Further investigations are warranted to determine the utility of KAEA to characterize mechanisms of disease progression and treatment failure using primary patient samples.

Effects of experimentally induced self-affirmation on the openness to meat reduction and alternative protein sources.

Introduction: Consumption of animals entails disregarding the pain of sentient beings, and acknowledging this can threaten an individual's image of oneself as a moral person. Also, abstaining from meat in a meat-eating culture can threaten an individual's valued group identity. Previous research on inter-group relations suggests that self-affirmation, affirmation of personally or collectively important values, can help individuals alleviate self-threats since it enhances one's global self-image and decreases threat perceptions. Methods: We tested for potential effects of self-affirmation on openness toward reducing meat consumption in an experimental study. Participants (N = 277) were randomized into an individual affirmation, group affirmation, or a control condition. Individual affirmation participants ranked a list of values and then wrote a short paragraph about their first-ranked value. Group affirmation participants did a similar task, focusing on the values of their ethnic group, while participants in the control condition had an unrelated task of ranking their color preferences. Participants then read a persuasive message presenting health risks related to meat consumption and the health benefits of reducing meat. Finally, they indicated their openness toward reducing meat consumption and acceptability of plant-based alternatives and lab-grown meat. Results and Discussion: Results show that affirmed participants expressed more readiness to reconsider their meat consumption habits, reduced perceptions of vegetarianism as a threat to the local culture, and more positive perceptions of the idea of lab-grown meat. However, self-esteem and frequency of meat consumption pose important limitations to the experimental effects. We discuss the findings from the perspective of self-and collective identity threats and the potential of self-affirmations to create a more open debate about animal product consumption.

Thrombin generation measurement using the ST Genesia Thrombin Generation System in a cohort of healthy adults: Normal values and variability.

BACKGROUND: Thrombin generation (TG) assays evaluate the balance between pro- and anticoagulant forces, to better assess bleeding and thrombotic risks. Although TG readouts obtained with the calibrated automated TG have been investigated in multiple clinical conditions, TG still needs standardization and clinical validation. The automated TG instrument ST Genesia® (STG, Stago, Asnières-sur-Seine, France) provides a normalization of TG parameters based on a reference plasma aiming to reduce the interlaboratory variability and the variability between different measurement runs. OBJECTIVES: To evaluate STG in a group of healthy adults. METHODS: Reference intervals in healthy adults and variability of the new standardized reagents for bleeding (BleedScreen) and thrombophilic (ThromboScreen) conditions were determined using STG. RESULTS: TG was measured in platelet-free plasma (PFP) samples of 123 healthy adults. Reference intervals were determined for TG parameters. Intra- and interassay coefficients of variation were calculated on quality controls and PFP samples from healthy adults. Oral contraception (OC) possibly influenced TG parameters, resulting in a higher median and a broader reference interval for peak height and endogenous thrombin potential (ETP) in women aged 20 to 49 years than in all other sex and age categories. Therefore, we propose the following reference interval categories: men, women aged <50 years not using OC, women aged <50 years using OC, and women aged ≥50 years. Normalization was effective to reduce the interassay variability of quality controls for ETP (BleedScreen assay), and peak height and ETP (ThromboScreen assay without thrombomodulin), but had little impact on PFP sample variability. CONCLUSION: STG appears suitable for accurate measurement of TG in healthy adults.

Semisolid self-microemulsifying drug delivery systems (SMEDDSs): Effects on pharmacokinetics of acyclovir in rats.

Semisolid self-microemulsifying drug delivery system (SMEDDS) with optimized drug loading capacity, stability, dispersibility in aqueous media and in vitro drug release profile, was evaluated in vivo regarding effects on pharmacokinetics of acyclovir, an antiviral with low bioavailability (BA) and short half-life (t1/2). Additional goal of this study was evaluation of safety of this semisolid SMEDDS consisted of medium chain length triglycerides (oil) (10% w/w), macrogolglycerol hydroxystearate (surfactant) (56.25% w/w), polyglyceryl-3-dioleate (cosurfactant) (6.25% w/w), glycerol (cosolvent) (20% w/w), macrogol 8000 (viscosity modifier) (7.5% w/w), and acyclovir (2.5 mg/ml). The study was performed on fully mature white male Wistar rats. The pharmacokinetics of acyclovir was monitored in three groups (1-3) of animals after administration of drug solution (intravenously (IV)), drug suspension (orally) and semisolid SMEDDS (orally), respectively. The determined pharmacokinetic parameters were: maximum concentration of acyclovir in serum (Cmax), time taken to reach Cmax (Tmax), areas under time-concentration curves (AUC0-t and AUC0-∞), terminal elimination rate constant (kel), t1/2, volume of distribution (Vd), mean residence time (MRT), clearance (Cl), zero concentration (C0), steady state volume of distribution (Vss), and BA. Additionally, for safety evaluation, animals were treated orally with aqueous solution of acyclovir, drug-free semisolid SMEDDS and acyclovir-loaded semisolid SMEDDS, during 21 days (groups 4-7). Serum samples of sacrificed animals were used for biochemical analysis of enzymatic activity of alanine transaminase (ALT) and aspartate transaminase (AST), urea, creatinine, and uric acid. Acyclovir administered by semisolid SMEDDS reached twice higher Cmax (0.92 ±â€¯0.21 µg/ml) and has significantly shorter Tmax (14 ±â€¯10.84 min) compared to the suspension of acyclovir (Cmax 0.29 ±â€¯0.09 µg/ml and Tmax 26.00 ±â€¯5.48 min). BA of the drug was significantly increased by semisolid SMEDDS, while the analysis of biochemical parameters excluded damage on function of liver and kidneys caused by the investigated drug delivery system.

Development of semisolid self-microemulsifying drug delivery systems (SMEDDSs) filled in hard capsules for oral delivery of aciclovir.

The study aimed to develop semisolid self-microemulsifying drug delivery systems (SMEDDSs) as carriers for oral delivery of aciclovir in hard hydroxypropylmethyl cellulose (HPMC) capsules. Six self-dispersing systems (SD1-SD6) were prepared by loading aciclovir into the semisolid formulations consisting of medium chain length triglycerides (lipid), macrogolglycerol hydroxystearate (surfactant), polyglyceryl-3-dioleate (cosurfactant), glycerol (hydrophilic cosolvent), and macrogol 8000 (viscosity modifier). Their characterization was performed in order to identify the semisolid system with rheological behaviour suitable for filling in hard HPMC capsules and fast dispersibility in acidic and alkaline aqueous media with formation of oil-in-water microemulsions. The optimal SMEDDS was loaded with aciclovir at two levels (2% and 33.33%) and morphology and aqueous dispersibility of the obtained systems were examined by applying light microscopy and photon correlation spectroscopy (PCS), respectively. The assessment of diffusivity of aciclovir from the SMEDDSs by using an enhancer cell model, showed that it was increased at a higher drug loading. Differential scanning calorimetry (DSC) analysis indicated that the SMEDDSs were semisolids at temperatures up to 50°C and physically stable and compatible with HPMC capsules for 3 months storage at 25°C and 4°C. The results of in vitro release study revealed that the designed solid dosage form based on the semisolid SMEDDS loaded with the therapeutic dose of 200mg, may control partitioning of the solubilized drug from in situ formed oil-in-water microemulsion carrier into the sorrounding aqueous media, and hence decrease the risk for precipitation of the drug.

Evaluation of critical formulation parameters in design and differentiation of self-microemulsifying drug delivery systems (SMEDDSs) for oral delivery of aciclovir.

The study investigated the influence of formulation parameters for design of self-microemulsifying drug delivery systems (SMEDDSs) comprising oil (medium chain triglycerides) (10%), surfactant (Labrasol(®), polysorbate 20, or Kolliphor(®) RH40), cosurfactant (Plurol(®) Oleique CC 497) (q.s. ad 100%), and cosolvent (glycerol or macrogol 400) (20% or 30%), and evaluate their potential as carriers for oral delivery of a poorly permeable antivirotic aciclovir (acyclovir). The drug loading capacity of the prepared formulations ranged from 0.18-31.66 mg/ml. Among a total of 60 formulations, three formulations meet the limits for average droplet size (Z-ave) and polydispersity index (PdI) that have been set for SMEDDSs (Z-ave≤100nm, PdI<0.250) upon spontaneous dispersion in 0.1M HCl and phosphate buffer pH 7.2. SMEDDSs with the highest aciclovir loading capacity (24.06 mg/ml and 21.12 mg/ml) provided the in vitro drug release rates of 0.325 mg cm(-2)min(-1) and 0.323 mg cm(-2)min(-1), respectively, and significantly enhanced drug permeability in the parallel artificial membrane permeability assay (PAMPA), in comparison with the pure drug substance. The results revealed that development of SMEDDSs with enhanced drug loading capacity and oral delivery potential, required optimization of hydrophilic ingredients, in terms of size of hydrophilic moiety of the surfactant, surfactant-to-cosurfactant mass ratio (Km), and log P of the cosolvent.