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Monitoring drug–target interactions with methods such as the cellular thermal-shift assay (CETSA) is well established for simple cell systems but remains challenging in vivo. Here we introduce tissue thermal proteome profiling (tissue-TPP), which measures binding of small-molecule drugs to proteins in tissue samples from drug-treated animals by detecting changes in protein thermal stability using quantitative mass spectrometry. We report organ-specific, proteome-wide thermal stability maps and derive target profiles of the non-covalent histone deacetylase inhibitor panobinostat in rat liver, lung, kidney and spleen and of the B-Raf inhibitor vemurafenib in mouse testis. In addition, we devised blood-CETSA and blood-TPP and applied it to measure target and off-target engagement of panobinostat and the BET family inhibitor JQ1 directly in whole blood. Blood-TPP analysis of panobinostat confirmed its binding to known targets and also revealed thermal stabilization of the zinc-finger transcription factor ZNF512. These methods will help to elucidate the mechanisms of drug action in vivo.The targets of small-molecule drugs are detected in tissue and blood using thermal proteome assays.

Background Titanium dioxide (TiO 2 ) is produced at high volumes and applied in many consumer and food products. Recent toxicokinetic modelling indicated the potential of TiO 2 to accumulate in human liver and spleen upon daily oral exposure, which is not routinely investigated in chronic animal studies. A health risk from nanosized TiO 2 particle consumption could not be excluded then. Results Here we show the first quantification of both total titanium (Ti) and TiO 2 particles in 15 post-mortem human livers and spleens. These low-level analyses were enabled by the use of fully validated (single particle) inductively coupled plasma high resolution mass spectrometry ((sp)ICP-HRMS) detection methods for total Ti and TiO 2 particles. The presence of TiO 2 in the particles in tissues was confirmed by Scanning Electron Microscopy with energy dispersive X-ray spectrometry. Conclusions These results prove that TiO 2 particles are present in human liver and spleen, with ≥24% of nanosize (< 100 nm). The levels are below the doses regarded as safe in animals, but half are above the dose that is deemed safe for liver damage in humans when taking into account several commonly applied uncertainty factors. With these new and unique human data, we remain with the conclusion that health risks due to oral exposure to TiO 2 cannot be excluded.

Nanomedicine is an emerging field with great potential in disease theranostics. We generated sterically stabilized superparamagnetic iron oxide nanoparticles (s-SPIONs) with average core diameters of 10 and 25 nm and determined the in vivo biodistribution and clearance profiles. Healthy nude mice underwent an intraperitoneal injection of these s-SPIONs at a dose of 90 mg Fe/kg body weight. Tissue iron biodistribution was monitored by atomic absorption spectroscopy and Prussian blue staining. Histopathological examination was performed to assess tissue toxicity. The 10 nm s-SPIONs resulted in higher tissue-iron levels, whereas the 25 nm s-SPIONs peaked earlier and cleared faster. Increased iron levels were detected in all organs and body fluids tested except for the brain, with notable increases in the liver, spleen, and the omentum. The tissue-iron returned to control or near control levels within 7 days post-injection, except in the omentum, which had the largest and most variable accumulation of s-SPIONs. No obvious tissue changes were noted although an influx of macrophages was observed in several tissues suggesting their involvement in s-SPION sequestration and clearance. These results demonstrate that the s-SPIONs do not degrade or aggregate in vivo and intraperitoneal administration is well tolerated, with a broad and transient biodistribution. In an ovarian tumor model, s-SPIONs were shown to accumulate in the tumors, highlighting their potential use as a chemotherapy delivery agent.

EBI2 receptors revealed as oxysterols The EBI2 receptor (Epstein–Barr virus-induced gene 2, also known as GPR183) was recently shown to be linked to autoimmune disease, and is a critical regulator of the humoral immune response. It is a G-protein-coupled receptor, and its natural ligand has been unknown. Two groups now bring an end to the 'orphan' status of this receptor with identification of specific oxysterols as its natural ligands. The most potent ligand and activator is 7a,25-dihydroxycholesterol, and the EBI2–oxysterol signalling pathway has an important role in the adaptive immune response. EBI2 (also called GPR183) is an orphan G-protein-coupled receptor that is highly expressed in spleen and upregulated upon Epstein–Barr-virus infection 1 . Recent studies indicated that this receptor controls follicular B-cell migration and T-cell-dependent antibody production 2 , 3 , 4 , 5 , 6 . Oxysterols elicit profound effects on immune and inflammatory responses as well as on cholesterol metabolism 7 , 8 , 9 . The biological effects of oxysterols have largely been credited to the activation of nuclear hormone receptors 10 , 11 . Here we isolate oxysterols from porcine spleen extracts and show that they are endogenous ligands for EBI2. The most potent ligand and activator is 7α,25-dihydroxycholesterol (OHC), with a dissociation constant of 450 pM for EBI2. In vitro , 7α,25-OHC stimulated the migration of EBI2-expressing mouse B and T cells with half-maximum effective concentration values around 500 pM, but had no effect on EBI2-deficient cells. In vivo , EBI2-deficient B cells or normal B cells desensitized by 7α,25-OHC pre-treatment showed reduced homing to follicular areas of the spleen. Blocking the synthesis of 7α,25-OHC in vivo with clotrimazole, a CYP7B1 inhibitor, reduced the content of 7α,25-OHC in the mouse spleen and promoted the migration of adoptively transferred pre-activated B cells to the T/B boundary (the boundary between the T-zone and B-zone in the spleen follicle), mimicking the phenotype of pre-activated B cells from EBI2-deficient mice. Our results show an unexpected causal link between EBI2, an orphan G-protein-coupled receptor controlling B-cell migration, and the known immunological effects of certain oxysterols, thus uncovering a previously unknown role for this class of molecules.

Active targeting of nanoparticles toward tumors is one of the most rapidly developing topics in nanomedicine. Typically, this strategy involves the addition of cancer-targeting biomolecules to nanoparticles, and studies on this topic have mainly focused on the localization of such formulations in tumors. Here, the analysis of the factors determining efficient nanoparticle targeting and therapy, various parameters such as types of targeting molecules, nanoparticle type, size, zeta potential, dose, and the circulation time are given. In addition, the important aspects such as how active targeting of nanoparticles alters biodistribution and how non-specific organ uptake influences tumor accumulation of the targeted nanoformulations are discussed. The analysis reveals that an increase in tumor accumulation of targeted nanoparticles is accompanied by a decrease in their uptake by the spleen. There is no association between targeting-induced changes of nanoparticle concentrations in tumors and other organs. The correlation between uptake in tumors and depletion in the spleen is significant for mice with intact immune systems in contrast to nude mice. Noticeably, modulation of splenic and tumor accumulation depends on the targeting molecules and nanoparticle type. The median survival increases with the targeting-induced nanoparticle accumulation in tumors; moreover, combinatorial targeting of nanoparticle drugs demonstrates higher treatment efficiencies. Results of the comprehensive analysis show optimal strategies to enhance the efficiency of actively targeted nanoparticle-based medicines.

Analyzing carbon monoxide concentration within an individual is crucial. The analysis of CO content in a tissue sample is performed using gas chromatography. The concentration is calculated based on a linear equation derived from the calibration curve created with the CO-fortified sample. However, when methemoglobin (MetHb) is formed from putrefaction, it inhibits CO binding to the sample and may lead to inaccurate results. MetHb results from the iron oxidation of normal heme hemoglobin (HHb), and by treating the sample with a reducing agent, it can be converted back to HHb. To investigate the effect of the reducing agent on spleen CO analysis, each sample was divided into two parts. One was treated with a 0.574 M sodium dithionite solution (Na 2 S 2 O 4 ), a reduced sample, and the other was treated with a rinse solution, serving as the control sample, with both undergoing the same preparation process and analyzed using Gas Chromatography with a Thermal Conductivity Detector (GC-TCD). Spleen samples from 60 autopsy cases were analyzed. The results indicated that 48 cases showed lower CO levels when the sample was reduced compared to the control sample, where the difference of the control and reduced samples ranged from 2.21 to 93.24%, with a median value of 13.83%. 12 cases exhibited no difference, where the difference between control and reduced sample ranged from 0.05 to 1.57%, with a median value of 0.67%. Our findings demonstrate that MetHb formed during decomposition can significantly inhibit CO binding in spleen tissue, leading to overestimation of CO levels when no reducing agent is used. Therefore, incorporating sodium dithionite treatment into GC-TCD methods improves the accuracy of postmortem CO quantification, particularly in putrefied samples.

The importance of small bioactive peptides derived from pig spleen have been used to enhance immune responses and support intestinal health. However, there is a lack of information regarding the conformational relationship and their effects on immune function of pig spleen proteins (PSPs). The objective of this study was to prepare and assess the immunomodulatory characteristics of immunopeptides from PSP. Firstly, enzymatic hydrolysates from PSP were prepared using alkaline protease and aminopeptidase, and small hydrolysate fractions with a <3 kDa were separated by SDS-PAGE and GPC. The bioactive peptides were then identified at peaks 5 to 7 (PSP-5, 6 and 7) by HPLC and TOF-MS, which were mainly composed of Pro-Glu-Leu by LC-MS. The PSP-5 and PSP-6 pronounced greater beneficial effects on cell viability and nitric oxide (NO) production than PSP-7 in macrophage, and PSP-5 exhibited a higher immunomodulatory ability than PSP-6. In vivo , the oral administration of 25-50 mg PSP-5/kg body weight (BW) protected against cyclophosphamide (CTX)-induced immunosuppression in spleen and intestine of mouse, as evidenced by increased cytokine and sIgA productions. In conclusion, a novel set of bioactive immunopeptides derived from PSP through enzymatic hydrolysis could enhance immunomodulatory properties.

Unlike mammalian red blood cells (RBCs), fish RBCs are nucleated and thus capable of gene expression. Japanese flounder (Paralichthys olivaceus) is a species of marine fish with important economic values. Flounder are susceptible to Edwardsiella tarda, a severe bacterial pathogen that is able to infect and survive in flounder phagocytes. However, the infectivity of and the immune response induced by E. tarda in flounder RBCs are unclear. In the present research, we found that E. tarda was able to invade and replicate inside flounder RBCs in both in vitro and in vivo infections. To investigate the immune response induced by E. tarda in RBCs, transcriptome analysis of the spleen RBCs of flounder challenged with E. tarda was performed. Six sequencing libraries were constructed, and an average of 43 million clean reads per library were obtained, with 85% of the reads being successfully mapped to the genome of flounder. A total of 1720 differentially expressed genes (DEGs) were identified in E. tarda-infected fish. The DEGs were significantly enriched in diverse Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, especially those associated with immunity, disease, and infection. Ninety-one key DEGs involved in 12 immune-related pathways were found to form extensive interaction networks. Twenty-one genes that constituted the hub of the networks were further identified, which were highly regulated by E. tarda and involved in a number of immune processes, notably pathogen recognition and signal transduction, antigen processing and presentation, inflammation, and splicing. These results provide new insights into the immune role of flounder RBCs during bacterial infection.

Delivery technologies are required for realizing the clinical potential of molecular medicines. This work presents an alternative technology to preformulated delivery systems by harnessing the natural transport properties of serum albumin using endogenous binding of gapmer antisense oligonucleotides (ASOs)/albumin constructs. We show by an electrophoretic mobility assay that fatty acid-modified gapmer and human serum albumin (HSA) can self-assemble into constructs that offer favorable pharmacokinetics. The interaction was dependent on fatty acid type (either palmitic or myristic acid), number, and position within the gapmer ASO sequence, as well as phosphorothioate (PS) backbone modifications. Binding correlated with increased blood circulation in mice (t1/2 increased from 23 to 49 min for phosphodiester [PO] gapmer ASOs and from 28 to 66 min for PS gapmer ASOs with 2× palmitic acid modification). Furthermore, a shift toward a broader biodistribution was detected for PS compared with PO gapmer ASOs. Inclusion of 2× palmitoyl to the ASOs shifted the biodistribution to resemble that of natural albumin. This work, therefore, presents a novel strategy based on the proposed endogenous assembly of gapmer ASOs/albumin constructs for increased circulatory half-life and modulation of the biodistribution of gapmer ASOs that offers tunable pharmacokinetics based on the gapmer modification design. Delivery technologies are required for realizing the clinical potential of molecular medicines. Howard et al. presents an alternative technology platform to preformulated delivery systems by harnessing the natural transport properties of serum albumin using endogenous binding of gapmer antisense oligonucleotides (ASOs)/albumin constructs tuned by chemical modifications.

Si/SiO x nanoparticles (NPs) produced by laser ablation in deionized water or aqueous biocompatible solutions present a novel extremely promising object for biomedical applications, but the interaction of these NPs with biological systems has not yet been systematically examined. Here, we present the first comprehensive study of biodistribution, biodegradability and toxicity of laser-synthesized Si-SiO x nanoparticles using a small animal model. Despite a relatively high dose of Si-NPs (20 mg/kg) administered intravenously in mice, all controlled parameters (serum, enzymatic, histological etc.) were found to be within safe limits 3 h, 24 h, 48 h and 7 days after the administration. We also determined that the nanoparticles are rapidly sequestered by the liver and spleen, then further biodegraded and directly eliminated in urine without any toxicity effects. Finally, we found that intracellular accumulation of Si-NPs does not induce any oxidative stress damage. Our results evidence a huge potential in using these safe and biodegradable NPs in biomedical applications, in particular as vectors, contrast agents and sensitizers in cancer therapy and diagnostics (theranostics).