Biophysical and structural characterization of the impacts of MET phosphorylation on tepotinib binding.

The receptor tyrosine kinase MET is activated by hepatocyte growth factor binding, followed by phosphorylation of the intracellular kinase domain (KD) mainly within the activation loop (A-loop) on Y1234 and Y1235. Dysregulation of MET can lead to both tumor growth and metastatic progression of cancer cells. Tepotinib is a highly selective, potent type Ib MET inhibitor and approved for treatment of non-small cell lung cancer harboring METex14 skipping alterations. Tepotinib binds to the ATP site of unphosphorylated MET with critical π-stacking contacts to Y1230 of the A-loop, resulting in a high residence time. In our study, we combined protein crystallography, biophysical methods (surface plasmon resonance, differential scanning fluorimetry), and mass spectrometry to clarify the impacts of A-loop conformation on tepotinib binding using different recombinant MET KD protein variants. We solved the first crystal structures of MET mutants Y1235D, Y1234E/1235E, and F1200I in complex with tepotinib. Our biophysical and structural data indicated a linkage between reduced residence times for tepotinib and modulation of A-loop conformation either by mutation (Y1235D), by affecting the overall Y1234/Y1235 phosphorylation status (L1195V and F1200I) or by disturbing critical π-stacking interactions with tepotinib (Y1230C). We corroborated these data with target engagement studies by fluorescence cross-correlation spectroscopy using KD constructs in cell lysates or full-length receptors from solubilized cellular membranes as WT or activated mutants (Y1235D and Y1234E/1235E). Collectively, our results provide further insight into the MET A-loop structural determinants that affect the binding of the selective inhibitor tepotinib.

Mesoporous Silica Nanoparticles Enhance the Anticancer Efficacy of Platinum(IV)-Phenolate Conjugates in Breast Cancer Cell Lines.

The main reasons for the limited clinical efficacy of the platinum(II)-based agent cisplatin include drug resistance and significant side effects. Due to their better stability, as well as the possibility to introduce biologically active ligands in their axial positions constructing multifunctional prodrugs, creating platinum(IV) complexes is a tempting strategy for addressing these limitations. Another strategy for developing chemotherapeutics with lower toxicity relies on the ability of nanoparticles to accumulate in greater quantities in tumor tissues through passive targeting. To combine the two approaches, three platinum(IV) conjugates based on a cisplatin scaffold containing in the axial positions derivatives of caffeic and ferulic acid were prepared and loaded into SBA-15 to produce the corresponding mesoporous silica nanoparticles (MSNs). The free platinum(IV) conjugates demonstrated higher or comparable activity with respect to cisplatin against different human breast cancer cell lines, while upon immobilization, superior antiproliferative activity with markedly increased cytotoxicity (more than 1000-fold lower IC50 values) compared to cisplatin was observed. Mechanistic investigations with the most potent conjugate, cisplatin-diacetyl caffeate (1), and the corresponding MSNs (SBA-15|1) in a 4T1 mouse breast cancer cell line showed that these compounds induce apoptotic cell death causing strong caspase activation. In vivo, in BALB/c mice, 1 and SBA-15|1 inhibited the tumor growth while decreasing the necrotic area and lowering the mitotic rate.

Anticancer Potential of Xanthohumol and Isoxanthohumol Loaded into SBA-15 Mesoporous Silica Particles against B16F10 Melanoma Cells.

Xanthohumol (XN) and isoxanthohumol (IXN), prenylated flavonoids from Humulus lupulus, have been shown to possess antitumor/cancerprotective, antioxidant, antiinflammatory, and antiangiogenic properties. In this study, mesoporous silica (SBA-15) was loaded with different amounts of xanthohumol and isoxanthohumol and characterized by standard analytical methods. The anticancer potential of XN and IXN loaded into SBA-15 has been evaluated against malignant mouse melanoma B16F10 cells. When these cells were treated with SBA-15 containing xanthohumol, an increase of the activity correlated with a higher immobilization rate of XN was observed. Considering the amount of XN loaded into SBA-15 (calculated from TGA), an improved antitumor potential of XN was observed (IC50 = 10.8 ± 0.4 and 11.8 ± 0.5 µM for SBA-15|XN2 and SBA-15|XN3, respectively; vs. IC50 = 18.5 ± 1.5 µM for free XN). The main mechanism against tumor cells of immobilized XN includes inhibition of proliferation and autophagic cell death. The MC50 values for SBA-15 loaded with isoxanthohumol were over 300 µg/mL in all cases investigated.

[About the Topic Hard of Hearing/Deafness in National and International Films]. / Über den Umgang mit dem Thema der Schwerhörigkeit/Gehörlosigkeit im nationalen und internationalen Fernseh- und Kinofilm.

Based on 415 movies which have been produced nationally and internationally and which the importance of the topic "hard of hearing and deafness" has been shown in TV as well as in cinemas are analyzed. Additionally, it has been summarized how real hard of hearing/deaf actors have been involved in such films. Categories of movies have been formed depending on the function of the fictive/real characters in the movies. The affiliation of the movies to different genres as well as how the number of films dealing with hard of hearing/deafness have been produced during the last decades has been analyzed. Topics which do interest especially the ENT-specialist are described. The estimation of the movies on the internet-based websites of "Internet Movie Database" and "Movie Pilot" given by the audience are presented.

Cisplatin-cyclooxygenase inhibitor conjugates, free and immobilised in mesoporous silica SBA-15, prove highly potent against triple-negative MDA-MB-468 breast cancer cell line.

For the development of anticancer drugs with higher activity and reduced toxicity, two approaches were combined: preparation of platinum(IV) complexes exhibiting higher stability compared to their platinum(II) counterparts and loading them into mesoporous silica SBA-15 with the aim to utilise the passive enhanced permeability and retention (EPR) effect of nanoparticles for accumulation in tumour tissues. Three conjugates based on a cisplatin scaffold bearing the anti-inflammatory drugs naproxen, ibuprofen or flurbiprofen in the axial positions (1, 2 and 3, respectively) were synthesised and loaded into SBA-15 to afford the mesoporous silica nanoparticles (MSNs) SBA-15|1, SBA-15|2 and SBA-15|3. Superior antiproliferative activity of both free and immobilised conjugates in a panel of four breast cancer cell lines (MDA-MB-468, HCC1937, MCF-7 and BT-474) with markedly increased cytotoxicity with respect to cisplatin was demonstrated. All compounds exhibit highest activity against the triple-negative cell line MDA-MB-468, with conjugate 1 being the most potent. However, against MCF-7 and BT-474 cell lines, the most notable improvement was found, with IC50 values up to 240-fold lower than cisplatin. Flow cytometry assays clearly show that all compounds induce apoptotic cell death elevating the levels of both early and late apoptotic cells. Furthermore, autophagy as well as formation of reactive oxygen species (ROS) and nitric oxide (NO) were elevated to a similar or greater extent than with cisplatin.

Synthesis, Crystallographic Structure, Theoretical Analysis, Molecular Docking Studies, and Biological Activity Evaluation of Binuclear Ru(II)-1-Naphthylhydrazine Complex.

Ruthenium(II)-arene complexes have gained significant research interest due to their possible application in cancer therapy. In this contribution two new complexes are described, namely [{RuCl(η6-p-cymene)}2(µ-Cl)(µ-1-N,N'-naphthyl)]X (X = Cl, 1; PF6, 2), which were fully characterized by IR, NMR, and elemental microanalysis. Furthermore, the structure of 2 in the solid state was determined by a single crystal X-ray crystallographic study, confirming the composition of the crystals as 2·2MeOH. The Hirshfeld surface analysis was employed for the investigation of interactions that govern the crystal structure of 2·2MeOH. The structural data for 2 out of 2·2MeOH was used for the theoretical analysis of the cationic part [{RuCl(η6-p-cymene)}2(µ-Cl)(µ-1-N,N'-naphthyl)]+ (2a) which is common to both 1 and 2. The density functional theory, at B3LYP/6-31+G(d,p) basis set for H, C, N, and Cl atoms and LanL2DZ for Ru ions, was used for the optimization of the 2a structure. The natural bond orbital and quantum theory of atoms in molecules analyses were employed to quantify the intramolecular interactions. The reproduction of experimental IR and NMR spectra proved the applicability of the chosen level of theory. The binding of 1 to bovine serum albumin was examined by spectrofluorimetry and molecular docking, with complementary results obtained. Compound 1 acted as a radical scavenger towards DPPH• and HO• radicals, along with high activity towards cancer prostate and colon cell lines.

Arene Ruthenium(II) Complexes Bearing the κ-P or κ-P,κ-S Ph2P(CH2)3SPh Ligand.

Neutral [Ru(η6-arene)Cl2{Ph2P(CH2)3SPh-κP}] (arene = benzene, indane, 1,2,3,4-tetrahydronaphthalene: 2a, 2c and 2d) and cationic [Ru(η6-arene)Cl(Ph2P(CH2)3SPh-κP,κS)]X complexes (arene = mesitylene, 1,4-dihydronaphthalene; X = Cl: 3b, 3e; arene = benzene, mesitylene, indane, 1,2,3,4-tetrahydronaphthalene, and 1,4-dihydronaphthalene; X = PF6: 4a-4e) complexes were prepared and characterized by elemental analysis, IR, 1H, 13C and 31P NMR spectroscopy and also by single-crystal X-ray diffraction analyses. The stability of the complexes has been investigated in DMSO. Complexes have been assessed for their cytotoxic activity against 518A2, 8505C, A253, MCF-7 and SW480 cell lines. Generally, complexes exhibited activity in the lower micromolar range; moreover, they are found to be more active than cisplatin. For the most active ruthenium(II) complex, 4b, bearing mesitylene as ligand, the mechanism of action against 8505C cisplatin resistant cell line was determined. Complex 4b induced apoptosis accompanied by caspase activation.

Discovery of Evobrutinib: An Oral, Potent, and Highly Selective, Covalent Bruton's Tyrosine Kinase (BTK) Inhibitor for the Treatment of Immunological Diseases.

Bruton's tyrosine kinase (BTK) inhibitors such as ibrutinib hold a prominent role in the treatment of B cell malignancies. However, further refinement is needed to this class of agents, particularly in terms of adverse events (potentially driven by kinase promiscuity), which preclude their evaluation in nononcology indications. Here, we report the discovery and preclinical characterization of evobrutinib, a potent, obligate covalent inhibitor with high kinase selectivity. Evobrutinib displayed sufficient preclinical pharmacokinetic and pharmacodynamic characteristics which allowed for in vivo evaluation in efficacy models. Moreover, the high selectivity of evobrutinib for BTK over epidermal growth factor receptor and other Tec family kinases suggested a low potential for off-target related adverse effects. Clinical investigation of evobrutinib is ongoing in several autoimmune diseases, including multiple sclerosis, rheumatoid arthritis, and systemic lupus erythematosus.

Impact of cell culture media additives on IgG glycosylation produced in Chinese hamster ovary cells.

Glycosylation is a key critical quality attribute for monoclonal antibodies and other recombinant proteins because of its impact on effector mechanisms and half-life. In this study, a variety of compounds were evaluated for their ability to modulate glycosylation profiles of recombinant monoclonal antibodies produced in Chinese hamster ovary cells. Compounds were supplemented into the cell culture feed of fed-batch experiments performed with a CHO K1 and a CHO DG44 cell line expressing a recombinant immunoglobulin G1 (IgG1). Experiments were performed in spin tubes or the ambr®15 controlled bioreactor system, and the impact of the compounds at various concentrations was determined by monitoring the glycosylation profile of the IgG and cell culture parameters, such as viable cell density, viability, and titer. Results indicate that the highest impact on mannosylation was achieved through 15 µM kifunensine supplementation leading to an 85.8% increase in high-mannose containing species. Fucosylation was reduced by 76.1% through addition of 800 µM 2-F-peracetyl fucose. An increase of 40.9% in galactosylated species was achieved through the addition of 120 mM galactose in combination with 48 µM manganese and 24 µM uridine. Furthermore, 6.9% increased sialylation was detected through the addition of 30 µM dexamethasone in combination with the same manganese, uridine, and galactose mixture used to increase total galactosylation. Further compounds or combinations of additives were also efficient at achieving a smaller overall glycosylation modulation, required, for instance, during the development of biosimilars. To the best of our knowledge, no evaluation of the efficacy of such a variety of compounds in the same cell culture system has been described. The studied cell culture media additives are efficient modulators of glycosylation and are thus a valuable tool to produce recombinant glycoproteins.

Spontaneous Isopeptide Bond Formation as a Powerful Tool for Engineering Site-Specific Antibody-Drug Conjugates.

Spontaneous isopeptide bond formation, a stabilizing posttranslational modification that can be found in gram-positive bacterial cell surface proteins, has previously been used to develop a peptide-peptide ligation technology that enables the polymerization of tagged-proteins catalyzed by SpyLigase. Here we adapted this technology to establish a novel modular antibody labeling approach which is based on isopeptide bond formation between two recognition peptides, SpyTag and KTag. Our labeling strategy allows the attachment of a reporting cargo of interest to an antibody scaffold by fusing it chemically to KTag, available via semi-automated solid-phase peptide synthesis (SPPS), while equipping the antibody with SpyTag. This strategy was successfully used to engineer site-specific antibody-drug conjugates (ADCs) that exhibit cytotoxicities in the subnanomolar range. Our approach may lead to a new class of antibody conjugates based on peptide-tags that have minimal effects on protein structure and function, thus expanding the toolbox of site-specific antibody conjugation.

S-Sulfocysteine simplifies fed-batch processes and increases the CHO specific productivity via anti-oxidant activity.

Industrial fed-batch cultivation of mammalian cells is used for the production of therapeutic proteins such as monoclonal antibodies. Besides medium ensuring initial growth, feeding is necessary to improve growth, viability and antibody production. Established processes include a slight acidic main feed and a separate alkaline feed containing l-tyrosine and l-cysteine. Since l-cysteine is not stable at neutral pH, a new derivative, S-sulfocysteine, was tested in neutral pH feeds. In small scale fed-batch processes, the S-sulfocysteine process yielded a comparable maximum viable cell density, prolonged viability and increased titer compared to the two feed system. Bioreactor experiments confirmed the increase in specific productivity. In depth characterization of the monoclonal antibody indicated no change in the glycosylation, or charge variant pattern whereas peptide mapping experiments were not able to detect any integration of the modified amino acid in the sequence of the monoclonal antibody. Finally, the mechanism of action of S-sulfocysteine was investigated, and results pointed out the anti-oxidative potential of the molecule, mediated through an increase in superoxide dismutase enzyme levels and in the total intracellular glutathione pool. Finally, we propose that the increase in specific productivity obtained in the S-sulfocysteine process results from the anti-oxidative properties of the molecule.

The development and design of the European Board of Otorhinolaryngology-Head and Neck Surgery Examination (EBEORL-HNS).

The UEMS Otorhinolaryngology-Head and Neck Surgery section is a dedicated body formed to promote the standardisation and harmonisation of European Otorhinolaryngology (ORL). The European Examination Board of Otorhinolaryngology and Head and Neck Surgery was created to establish a supranational final exam and accreditation for ORL Surgeons. It is open to candidates both from the European Union and outside the EU. The exam is composed of a written examination to assess mainly the theoretical knowledge of Otorhinolaryngological diseases. The second part, a viva voce examination, is designed to test the clinical application of knowledge based on case scenarios and clinical conditions presented to the candidates. The inaugural examination written component took place in Mannheim/Germany in 2009 and the inaugural Viva Voce examination in Vienna/Austria in 2010. Up to and including the year 2013, 858 participants have attempted one of the two exam components. Of the 858 participants, 305 were successful in both examinations and obtained the accreditation of the European Diploma (European Board Certification). The historical origins, development of the examination, its formal arrangements and the format of the examination are presented in this article.

Global surgery in a postconflict setting--5-year results of implementation in the Russian North Caucasus.

BACKGROUND: Collaborations for global surgery face many challenges to achieve fair and safe patient care and to build sustainable capacity. The 2004 terrorist attack on a school in Beslan in North Ossetia in the Russian North Caucasus left many victims with complex otologic barotrauma. In response, we implemented a global surgery partnership between the Vladikavkaz Children's Hospital, international surgical teams, the North Ossetian Health Ministry, and civil society organizations. This study's aim was to describe the implementation and 5-year results of capacity building for complex surgery in a postconflict, mid-income setting. DESIGN: We conducted an observational study at the Children's Hospital in Vladikavkaz in the autonomous Republic of North Ossetia-Alania, part of the Russian Federation. We assessed the outcomes of 15 initial patients who received otologic surgeries for complex barotrauma resulting from the Beslan terrorism attack and for other indications, and report the incidence of intra- and postoperative complications. RESULTS: Patients were treated for trauma related to terrorism (53%) and for indications not related to violence (47%). None of the patients developed peri- or postoperative complications. Three patients (two victims of terrorism) who underwent repair of tympanic perforations presented with re-perforations. Four junior and senior surgeons were trained on-site and in Germany to perform and teach similar procedures autonomously. CONCLUSIONS: In mid-income, postconflict settings, complex surgery can be safely implemented and achieve patient outcomes comparable to global standards. Capacity building can build on existing resources, such as operation room management, nursing, and anesthesia services. In postconflict environments, substantial surgical burden is not directly attributable to conflict-related injury and disease, but to health systems weakened by conflicts. Extending training and safe surgical care to include specialized interventions such as microsurgery are integral components to strengthen local capacity and ownership. Our experience identified strategies for fair patient selection and might provide a model for potentially sustainable surgical system building in postconflict environments.

In vitro effects of binuclear (η (6)-p-cymene)ruthenium(II) complex containing bridging bis(nicotinate)-polyethylene glycol ester ligand on differentiation pathways of murine Th lymphocytes activated by T cell mitogen.

T cell differentiation into distinct T helper (Th) subpopulations is crucial in governing acquired immune responses as well as some inflammatory and autoimmune disorders. This study investigated potential of the novel neutral binuclear ruthenium(II) complexes 1-8 with general formula [{RuCl2(η(6)-p-cym)}2µ-(N(∩)N)] (N(∩)N = bis(nicotinate)- and bis(iso-nicotinate)-polyethylene glycol esters; (3-py)COO(CH2CH2O) n CO(3-py) and (4-py)COO(CH2CH2O) n CO(4-py); n = 1-4), as well as [RuCl2(η(6)-p-cym)(nic)] (R1, nic = nicotinate) and [RuCl2(η(6)-p-cym)(inic)] (R2, inic = isonicotinate) as an immunomodulatory agents capable to direct Th cell differentiation. From all investigated complexes, [{RuCl2(η(6)-p-cym)}2µ-{(3-py)COO(CH2CH2O)4CO(3-py)}] (4) was selected for further study because it did not affect splenocyte viability (in concentration up to 50 µM), but significantly reduced secretion of representative Th1 cytokine, IFN-γ induced by T cell mitogen. Besides IFN-γ, 4 inhibited dose dependently expression and production of representative Th17 cytokine, IL-17, in these cells. Otherwise, the production of anti-inflammatory cytokines IL-4 and IL-10 was upregulated. Also, 4 significantly increased CD4(+)CD25(+)FoxP3(+) Treg cell frequency in the activated splenocytes. Moreover, ConA-induced expression of Th1 transcription factors, T-bet and STAT1, as well as of Th17-related protein STAT3 was attenuated upon exposure to 4, while the expression of Th2-related transcription factor GATA3 remained stable. In conclusion, ruthenium(II) complex 4 modulates immune system cell functions in vitro by inhibiting T cell differentiation towards pathogenic Th1/Th17 phenotype and inducing a regulatory phenotype characterized by IL-10 and IL-4 production, which may provide novel therapeutic opportunities for immune-inflammatory and/or autoimmune disorders.

Quantitative site-specific analysis of protein glycosylation by LC-MS using different glycopeptide-enrichment strategies.

A common technique for analysis of protein glycosylation is HPLC coupled to mass spectrometry (LC-MS). However, analysis is challenging due to a low abundance of glycopeptides in complex protein digests, microheterogeneity at the glycosylation site, ion suppression effects, and competition for ionization by coeluting peptides. Specific sample preparation is necessary for a comprehensive and site-specific glycosylation analysis by MS. In this study we qualitatively compared hydrophilic interaction chromatography (HILIC) and hydrazine chemistry for the enrichment of all N-linked glycopeptides and titanium dioxide for capturing sialylated glycopeptides from a complex peptide mixture. Bare silica, microcrystalline cellulose, amino-, amide- (TSKgel Amide-80), and sulfobetaine-(ZIC-HILIC) bonded phases were evaluated for HILIC enrichment. The experiments revealed that ZIC-HILIC and TSKgel Amide-80 are very specific for capturing glycopeptides under optimized conditions. Quantitative analysis of N-glycosidase F-released and 2-aminobenzamide-labeled glycans of a ZIC-HILIC-enriched monoclonal antibody demonstrated that glycopeptides could be enriched without bias for particular glycan structures and without significant losses. Sialylated glycopeptides could be efficiently enriched by titanium dioxide and in addition to HILIC both methods enable a comprehensive analysis of protein glycosylation by MS. Enrichment of N-linked glycopeptides by hydrazine chemistry resulted in lower peptide recovery using a more complex enrichment scheme.