Fragmentation of a dioxolanyl radical via nonstatistical reaction dynamics: characterization of the vinyloxy radical by ns time-resolved laser flash photolysis

The photochemistry of two Barton esters, one derived from a dioxolane carboxylic acid and the other from pivalic acid, was investigated by product analysis and nanosecond laser flash photolysis (LFP). As expected, photolysis of the pivalate ester resulted in formation of the pyridine-2-thiyl and the t-butyl radical. Photolysis of the Barton ester of 2,2-dimethyl-1,3-dioxolane-4-carboxylic acid, on the other hand, revealed a complex multi-step fragmentation. In addition to the pyridine-2-thiyl and dioxolanyl radical, we gained evidence for the formation of the vinyloxy radical, CH2[double bond, length as m-dash]CHO˙. The latter was identified in the LFP by its π-complexes with benzene and diphenylether, its rapid quenching by electron-rich arenes and tri-n-butyl tin hydride, and its oxidative power in presence of trifluoroacetic acid as demonstrated by the oxidation of ferrocene to ferrocenium. Formation of CH2[double bond, length as m-dash]CHO˙ can be rationalized via fragmentation of the dioxolanyl radical. As the calculated barriers are too high for the reaction sequence to occur on the LFP time scale, we investigated the fragmentation of the photoexcited Barton ester via Born–Oppenheimer molecular dynamics simulations. In one trajectory, we could observe all reaction steps including ring opening of the dioxolanyl radical, suggesting that the excess energy gained in the ester cleavage and decarboxylation may lead to fragmentation of the hot dioxolanyl radical

Treatment of uveal melanoma

Titelblatt Einleitung Entwicklung adjuvanter Therapieansätze Systemische Therapieansätze bei metastasierter Erkrankung Diskussion Danksagung Literatur ErklärungDas Aderhautmelanom hat je nach Risikokonstellation zum Zeitpunkt der Erstdiagnose ein Metastasierungsrisiko von bis zu 70% innerhalb von 5 Jahren. Die Entwicklung effektiver adjuvanter Therapiestrategien mit dem Ziel der Reduktion des Metastasierungsrisikos und der Verbesserung des Überlebens ist eine der wichtigsten Herausforderungen. Durch die rasante Entwicklung der Tumorimmunologie in den letzten Jahren ist es gelungen in der Klinik erste tumorspezifische Vakzinierungsstudien durchzuführen. Die Etablierung und Evaluation des ELISPOT Assays erlaubt die ex vivo Quantifizierung spezifischer T Lymphozyten im Rahmen von Vakzinierungsstudien und eröffnet somit die Möglichkeit Vakzinierungsansätze weiter zu optimieren. So konnte gezeigt werden, dass Tumorvakzinierungen bei einem Teil der Patienten mit hohem Metastasierungsrisiko wirksam sind. In den nächsten Jahren werden solche Therapieprinzipien in der adjuvanten Therapie des Aderhautmelanoms weiterzuentwickeln sein. Die Prognose von Patienten mit metastasierter Erkrankung ist außerordentlich schlecht. In der palliativen Situation gelang es, basierend auf in-vitro Untersuchungen zur Chemotherapieresistenz, eine Kombinationstherapie aus Gemcitabin und Treosulfan zu entwickeln und einen in- vitro postulierten Synergismus beider Substanzen in-vivo zu bestätigen. Die Frage, ob eine lokoregionäre Chemotherapie über die Arteria hepatica möglicherweise einer systemischen, intravenösen Chemotherapie überlegen ist, wird derzeit in einer multizentrischen Studie untersucht.The risk of metastases from uveal melanoma depending on individual risk factors is 70% in 5 years. Development of effective adjuvant treatment approaches aiming at the reduction of the risk of metastases and at an improvement of survival is crucial. The rapid developments in the field of tumor immunology during recent years resulted in tumorspecific vaccination trials. The establishment and evaluation of the ELISPOT assay allowed improvement of vaccination strategies. It could be demonstrated that tumorspecific vaccination might be effective in patients with high risk of metastatic relapse. In the near future such vaccination approaches should be further evaluated in the adjuvant treatment of high risk uveal melanoma. Prognosis of patients with metastatic uveal melanoma is poor. Based on in- vitro chemotherapy sensitivity assays a combination chemotherapy consisting of gemcitabine and treosulfan could be established and in vitro synergism could be confirmed in-vivo. Wheather loco-regional therapy via the hepatic artery might be superior to intravenous chemotherapy is currently investigated in a multicenter trial

Spectroscopic and theoretical approaches for studying radical reactions in class I ribonucleotide reductase

Ribonucleotide reductases (RNRs) catalyze the production of deoxyribonucleotides, which are essential for DNA synthesis and repair in all organisms. The three currently known classes of RNRs are postulated to utilize a similar mechanism for ribonucleotide reduction via a transient thiyl radical, but they differ in the way this radical is generated. Class I RNR, found in all eukaryotic organisms and in some eubacteria and viruses, employs a diferric iron center and a stable tyrosyl radical in a second protein subunit, R2, to drive thiyl radical generation near the substrate binding site in subunit R1. From extensive experimental and theoretical research during the last decades, a general mechanistic model for class I RNR has emerged, showing three major mechanistic steps: generation of the tyrosyl radical by the diiron center in subunit R2, radical transfer to generate the proposed thiyl radical near the substrate bound in subunit R1, and finally catalytic reduction of the bound ribonucleotide. Amino acid- or substrate-derived radicals are involved in all three major reactions. This article summarizes the present mechanistic picture of class I RNR and highlights experimental and theoretical approaches that have contributed to our current understanding of this important class of radical enzymes

High background in ELISpot assays is associated with elevated levels of immune activation in HIV‐1‐seronegative individuals in Nairobi

Introduction: Spontaneous interferon‐γ (IFNγ) released detected by enzyme‐linked immunospot (ELISpot) assays may be a biological phenomenon. Markers of immune activation levels were assessed as correlates of high background among individuals in Kenya. Methods: Couples concordantly seronegative for HIV‐1 were enrolled. IFN‐γ ELISpot assays were conducted and negative control wells were categorized as having either high or low background (≥50 and \u3c50 SFU/106 peripheral blood mononuclear cells [PBMC], respectively). PBMC were stained for CD4, CD8, and immune activation markers (CD38 and HLA‐DR) and analyzed using flow cytometry. Proportions of activated T‐cells were compared between those with low and high background by Mann–Whitney U test. Correlates of background SFU and immune activation were assessed using regression models. Results: Among 58 individuals, 14 (24%) had high background. Frequencies of CD4+CD38+HLA‐DR+ and CD8+CD38+HLA‐DR+ cells were higher in individuals with high background compared to those with low background (P = 0.02). Higher background SFU was associated with history of sexually transmitted infections (P = 0.03), and illness in the past 3 months (P = 0.005), in addition to increased levels of activated CD4+ and CD8+ cells (P range = 0.008–0.03). Female gender and male circumcision decreased levels of CD4+ and CD8+ immune activation (P range = 0.002–0.03). Additionally, higher background SFU and activated CD4+ and CD8+ cells were individually associated with positive ELISpot responses to HIV‐1 peptide pools (P range = 0.01–0.03). Conclusions: These findings suggest that increased basal immune responses may be a biological mechanism contributing to higher background ELISpot SFU. Systematic exclusion of data from individuals with increased background in IFN‐γ release assays may bias results in population‐based studies

Negative enrichment by immunomagnetic nanobeads for unbiased characterization of circulating tumor cells from peripheral blood of cancer patients

BACKGROUND: A limitation of positive selection strategies to enrich for circulating tumor cells (CTCs) is that there might be CTCs with insufficient expression of the surface target marker which may be missed by the procedure. We optimized a method for enrichment, subsequent detection and characterization of CTCs based on depletion of the leukocyte fraction. METHODS: The 2-step protocol was developed for processing 20 mL blood and based on red blood cell lysis followed by leukocyte depletion. The remaining material was stained with the epithelial markers EpCAM and cytokeratin (CK) 7/8 or for the melanoma marker HMW-MAA/MCSP. CTCs were detected by flow cytometry. CTCs enriched from blood of patients with carcinoma were defined as EpCAM+CK+CD45-. CTCs enriched from blood of patients with melanoma were defined as MCSP+CD45-. One-hundred-sixteen consecutive blood samples from 70 patients with metastatic carcinomas (n = 48) or metastatic melanoma (n = 22) were analyzed. RESULTS: CTCs were detected in 47 of 84 blood samples (56%) drawn from carcinoma patients, and in 17 of 32 samples (53%) from melanoma patients. CD45-EpCAM-CK+ was detected in pleural effusion specimens, as well as in peripheral blood samples of patients with NSCLC. EpCAM-CK+ cells have been successfully cultured and passaged longer than six months suggesting their neoplastic origin. This was confirmed by CGH. By defining CTCs in carcinoma patients as CD45-CK+ and/or EpCAM+, the detection rate increased to 73% (61/84). CONCLUSION: Enriching CTCs using CD45 depletion allowed for detection of epithelial cancer cells not displaying the classical phenotype. This potentially leads to a more accurate estimation of the number of CTCs. If detection of CTCs without a classical epithelial phenotype has clinical relevance need to be determined

Raf-1 kinase associates with Hepatitis C virus NS5A and regulates viral replication

AbstractHepatitis C virus (HCV) is a positive-strand RNA virus that frequently causes persistent infection associated with severe liver disease. HCV nonstructural protein 5A (NS5A) is essential for viral replication. Here, the kinase Raf-1 was identified as a novel cellular binding partner of NS5A, binding to the C-terminal domain of NS5A. Raf-1 colocalizes with NS5A in the HCV replication complex. The interaction of NS5A with Raf-1 results in increased Raf-1 phosphorylation at serine 338. Integrity of Raf-1 is crucial for HCV replication: inhibition of Raf-1 by the small-molecule inhibitor BAY43-9006 or downregulation of Raf-1 by siRNA attenuates viral replication

FATORES QUE AFETAM A PERCEPÇÃO DO COLABORADOR SOBRE A ORIENTAÇÃO PARA O MERCADO DE UMA EMPRESA DO SETOR DE TECNOLOGIA DA INFORMAÇÃO

Orientação para o mercado é a capacidade que uma organização tem de entender e satisfazer seus clientes a partir da identificação das suas necessidades e adequação dos produtos e serviços ofertados. Tal capacidade gera benefícios como aumento das vendas, lucratividade, faturamento e satisfação dos funcionários. O objetivo desta pesquisa foi identificar junto aos colaboradores de uma empresa de tecnologia da informação os fatores que, na percepção deles, afetam a orientação para o mercado da empresa. Foi feita uma pesquisa quantitativa, descritiva, com corte transversal, por meio de questionários respondidos por 177 funcionários de uma empresa do setor, nos quais foram gerados dados posteriormente analisados por regressão linear múltipla. Após a análise, constatou-se, na opinião dos funcionários, que a empresa pode obter melhor orientação para o mercado se ouvir os anseios e as reclamações dos clientes, fazer pesquisas de mercado, manter um nível adequado de comunicação interna e desenvolver produtos e serviços alinhados com o que o cliente quer