Vemurafenib and Dabrafenib Downregulates RIPK4 Level.

Vemurafenib and dabrafenib are BRAF kinase inhibitors (BRAFi) used for the treatment of patients with melanoma carrying the V600E BRAF mutation. However, melanoma cells develop resistance to both drugs when used as monotherapy. Therefore, mechanisms of drug resistance are investigated, and new molecular targets are sought that could completely inhibit melanoma progression. Since receptor-interacting protein kinase (RIPK4) probably functions as an oncogene in melanoma and its structure is similar to the BRAF protein, we analyzed the impact of vemurafenib and dabrafenib on RIPK4 in melanomas. The in silico study confirmed the high similarity of BRAF kinase domains to the RIPK4 protein at both the sequence and structural levels and suggests that BRAFi could directly bind to RIPK4 even more strongly than to ATP. Furthermore, BRAFi inhibited ERK1/2 activity and lowered RIPK4 protein levels in BRAF-mutated melanoma cells (A375 and WM266.4), while in wild-type BRAF cells (BLM and LoVo), both inhibitors decreased the level of RIPK4 and enhanced ERK1/2 activity. The phosphorylation of phosphatidylethanolamine binding protein 1 (PEBP1)-a suppressor of the BRAF/MEK/ERK pathway-via RIPK4 observed in pancreatic cancer did not occur in melanoma. Neither downregulation nor upregulation of RIPK4 in BRAF- mutated cells affected PEBP1 levels or the BRAF/MEK/ERK pathway. The downregulation of RIPK4 inhibited cell proliferation and the FAK/AKT pathway, and increased BRAFi efficiency in WM266.4 cells. However, the silencing of RIPK4 did not induce apoptosis or necroptosis. Our study suggests that RIPK4 may be an off-target for BRAF inhibitors.

Lutein and Zeaxanthin in the Lipid Bilayer-Similarities and Differences Revealed by Computational Studies.

Lutein and zeaxanthin are two similar carotenoids of the xanthophyll subgroup. Carotenoids are synthesized almost entirely by plants but are also present in significant amounts in animals. They are essential components of the lipid matrix of biomembranes, and one of their functions is to protect cells from light radiation, free radicals and oxidative stress. Carotenoids, depending on their chemical structure, can locate at various positions and in different orientations in the bilayer. Xanthophylls (XAN) are polar and in the bilayer are positionally restricted. In the case of lutein and zeaxanthin, whose both ionone rings are hydroxy-substituted and as such are anchored in the lipid bilayer interfaces, the position is generally transmembrane. However, both experimental and computer modelling studies indicate that lutein can also locate horizontally below the bilayer interface. This location has never been observed for zeaxanthin. To find a molecular-level explanation for the difference in the orientations of the XAN molecules in the bilayer, a number of phosphatidylcholine-XAN bilayers were constructed and molecular dynamics (MD) simulated for 1.1 µs each. The all-trans XAN molecules were initially placed either parallel or perpendicular to the bilayer surface. With the exception of one lutein, the horizontally placed molecules adopted the transmembrane orientation within 100-600 ns. On the basis of detailed analyses of the XAN orientations and the numbers and lifetimes of their interactions in the bilayer, a plausible explanation is offered as to why a lutein molecule may remain in the horizontal orientation while zeaxanthin does not. Contrary to common believe, lutein horizontal orientation is not related to the ε-ring rotation around the C6'-C7' bond.

Asymmetric Spontaneous Intercalation of Lutein into a Phospholipid Bilayer, a Computational Study.

Lutein, a hydroxylated carotenoid, is a pigment synthesised by plants and bacteria. Animals are unable to synthesise lutein, nevertheless, it is present in animal tissues, where its only source is dietary intake. Both in plants and animals, carotenoids are associated mainly with membranes where they carry out important physiological functions. Their trafficking to and insertion into membranes are not well recognised due to experimental difficulties. In this paper, a computational approach is used to elucidate details of the dynamics and energetics of lutein intercalation from the water to the phospholipid bilayer phase. The dynamics is studied using molecular dynamics simulation, and the energetics using umbrella sampling. Lutein spontaneous insertion into the bilayer and translocation across it proceed via formation of hydrogen bonds between its hydroxyl groups and water and/or phospholipid oxygen atoms as well as desolvation of its polyene chain. As lutein molecule is asymmetric, its bilayer intercalation is also asymmetric. The course of events and timescale of the intercalation are different from those of helical peptides. The time of full lutein intercalation ranges from 20 to 100 ns and its final orientation is predominately vertical. Nevertheless, some lutein molecules are in the final horizontal position and some aggregate in the water phase and remain there for the whole simulation time. The highest energy barrier for the intercalation process is ~2.2 kcal/mol and the energy gain is ~18 kcal/mol. The results obtained for lutein can be applied to other xanthophylls and molecules of a similar structure.

Usefulness of laser ablation ICP-MS for analysis of metallic particles released to oral mucosa after insertion of dental implants.

Despite the fact that titanium is considered highly biocompatible, its presence in the oral cavity (an environment of frequently changing pH and temperature) may result in the release of titanium from intraosseous implants into the oral mucosa, causing a range of reactions from the human body. Fragments of oral mucosa collected from patients after dental implant insertion were analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The study revealed an elevated content of elements (Ti, Al, V) which are components of the metal implants and temporary cover screws. Dynamic ablation of the tissue surface was used in order to obtain maps of the content and distribution of analyzed elements. The material consisted of 30 oral mucosa tissue fragments collected 3-5 months after implantation and 10 samples collected before implantation (control group). The application of optical microscope allowed for indication and confirmation of the location of metal particles prior to LA-ICP-MS analysis. The so-obtained map permitted location of regions containing metal particles. LA-ICP-MS analysis revealed groups of samples with similar properties of metal particles, thus confirming that those metal particles were the main source of the elevated content of metals (Ti, Al, V) in the tissue after implantation. A calibration strategy based on matrix matched solid standards with powdered egg white proteins as matrix material was applied with 34S as an internal standard. The accuracy of the analytical method was verified by ablating pellets of certified reference material ERM-BB422 Fish muscle.

Agglutinating mouse IgG3 compares favourably with IgMs in typing of the blood group B antigen: Functionality and stability studies.

Mouse immunoglobulins M (IgMs) that recognize human blood group antigens induce haemagglutination and are used worldwide for diagnostic blood typing. Contrary to the current belief that IgGs are too small to simultaneously bind antigens on two different erythrocytes, we obtained agglutinating mouse IgG3 that recognized antigen B of the human ABO blood group system. Mouse IgG3 is an intriguing isotype that has the ability to form Fc-dependent oligomers. However, F(ab')2 fragments of the IgG3 were sufficient to agglutinate type B red blood cells; therefore, IgG3-triggered agglutination did not require oligomerization. Molecular modelling indicated that mouse IgG3 has a larger range of Fab arms than other mouse IgG subclasses and that the unique properties of mouse IgG3 are likely due to the structure of its hinge region. With a focus on applications in diagnostics, we compared the stability of IgG3 and two IgMs in formulated blood typing reagents using an accelerated storage approach and differential scanning calorimetry. IgG3 was much more stable than IgMs. Interestingly, the rapid decrease in IgM activity was caused by aggregation of the molecules and a previously unknown posttranslational proteolytic processing of the µ heavy chain. Our data point to mouse IgG3 as a potent diagnostic tool.