SNPase-ARMS qPCR: Ultrasensitive Mutation-Based Detection of Cell-Free Tumor DNA in Melanoma Patients

Cell-free circulating tumor DNA in the plasma of cancer patients has become a common point of interest as indicator of therapy options and treatment response in clinical cancer research. Especially patient- and tumor-specific single nucleotide variants that accurately distinguish tumor DNA from wild type DNA are promising targets. The reliable detection and quantification of these single-base DNA variants is technically challenging. Currently, a variety of techniques is applied, with no apparent “gold standard”. Here we present a novel qPCR protocol that meets the conditions of extreme sensitivity and specificity that are required for detection and quantification of tumor DNA. By consecutive application of two polymerases, one of them designed for extreme base-specificity, the method reaches unprecedented sensitivity and specificity. Three qPCR assays were tested with spike-in experiments, specific for point mutations BRAF V600E, PTEN T167A and NRAS Q61L of melanoma cell lines. It was possible to detect down to one copy of tumor DNA per reaction (Poisson distribution), at a background of up to 200 000 wild type DNAs. To prove its clinical applicability, the method was successfully tested on a small cohort of BRAF V600E positive melanoma patients.     

Multicolour Fluorescence-Detection Size-Exclusion Chromatography for Structural Genomics of Membrane Multiprotein Complexes

Many interesting and important membrane proteins are hetero-oligomeric. However, besides naturally abundant examples, the structures of relatively few such complexes are known. Partly, this is due to difficulties in expression, stoichiometric assembly, and in the evaluation of their stability prior to crystallization trials. Here we describe a new approach, which allows rapid assessment of protein complex quality, assembly and stoichiometry, simplifying the search for conditions conducive to long-term stability and crystallization. Multicolour fluorescence size-exclusion chromatography (MC-FSEC) is used to enable tracking of individual subunits through expression, solubilization and purification steps. We show how the method has been applied to the heterodimeric transporter associated with antigen processing (TAP) and demonstrate how it may be extended in order to analyse membrane multisubunit assemblies.     

Responses of solitary and colonial coronate polyps (Cnidaria, Scyphozoa, Coronatae) to sedimentation and burial

Coronate polyps retract their soft bodies into a protective peridermal tube after mechanical irritation. Sediment may enter the tube of contracted polyps, however, and block the opening [Jarms, G., 1990. Neubeschreibung dreier Arten der Gattung Nausithoe (Coronatae, Scyphozoa) sowie Wiederbeschreibung der Art Nausithoe marginata Kölliker, 1853. Mitt. Hamb. Zool. Mus. Inst. 87, 7-39; Silveira, F.L. da, Jarms, G., Morandini, A.C., 2003. Experiments in nature and laboratory observations with Nausithoe aurea (Scyphozoa: Coronatae) support the concept of perennation by tissue saving and confirm dormancy. Biota Neotropica 2 (2), 1-25]. In the present study, the ability of different coronate species [Nausithoe aurea Silveira and Morandini, 1997, Nausithoe planulophora (Werner, 1971), Thecoscyphus zibrowii Werner, 1984, Linuche unguiculata (Swarts, 1788)] to expel sediment particles from the tube was investigated. In laboratory experiments, sand grains and mussel shell fragments were inserted into the tubes and the responses of polyps were observed. Particles were ingested as polyps extended themselves, and after extension, they were defecated. Ingestion was effected by an aborally directed flagellar beat of the flagellated gastrodermal epithelia that was reversed for defecation. Particles only slightly smaller than the tube opening could be expelled, and extension of the polyp was possible even if grains blocked 2/3 of the tube. However, if particles became stuck in the tube, ingestion was impossible and polyp extension failed. Comparisons among the tested species showed that expulsion success depended on tube shape and polyp morphology. In N. aurea and N. planulophora, less than 5% of tubes were permanently blocked. The cave-dwelling species T. zibrowii was not able to ingest particles due to its particular morphology, and in 25% of experiments with shell fragments tubes were permanently blocked. Thin, elongate tubes of the colonial polyps L. unguiculata were also often permanently blocked by shell fragments (50%), but new polyps were developed from the scyphorhiza to ensure survival of the colony. Solitary polyps were able to survive more than 5 months retracted beneath any blocking particles. After tubes were cut off beneath such particles normal polyps developed. From our observations, we suggest that coronate polyps can exist in habitats with moderate sedimentation, and that they can survive being temporarily buried.     

Early cortical facilitation for emotionally arousing targets during the attentional blink

Background  

The present study aimed to investigate the time course of electrocortical facilitation for affectively arousing written words during the so-called 'attentional blink' (AB) period in a rapid serial visual presentation (RSVP) task. The AB refers to a period of reduced awareness for second-target stimuli following a first target by an interval of about 200–500 ms. Pleasant, neutral, and unpleasant written verbs were used as second targets in an 8.6-Hz RSVP paradigm that contained affectively neutral words as distractors. Replicating and extending behavioral studies, we expected that emotional second targets would be associated with better identification accuracy and greater electrocortical activity, compared with neutral targets.     

Checkpoint kinase 1 negatively regulates somatic hypermutation

Immunoglobulin (Ig) diversification by somatic hypermutation in germinal center B cells is instrumental for maturation of the humoral immune response, but also bears the risk of excessive or aberrant genetic changes. Thus, introduction of DNA damage by activation-induced cytidine deaminase as well as DNA repair by multiple pathways need to be tightly regulated during the germinal center response to prevent lymphomagenesis. In the present study, we show that DNA damage checkpoint signaling via checkpoint kinase 1 (Chk1) negatively regulates somatic hypermutation. Chk1 inhibition in human B cell lymphoma lines as well as inactivation of Chk1 alleles by gene targeting in DT40 B cells leads to increased somatic hypermutation. This is apparently due to changes in DNA repair pathways regulated by Chk1, such as a decreased homologous recombination efficiency that also leads to decreased Ig gene conversion in DT40. Our data show that Chk1 signaling plays a crucial role in regulation of Ig diversification and sheds unexpected light on potential origins of aberrant somatic hypermutation in B cell lymphomagenesis.     

Selection and characterization of a human monoclonal neutralizing antibody for Clostridium Botulinum neurotoxin serotype B

Botulinum neurotoxins (BoNTs) are causative agents for botulism and are identified as a category A bioterror agents by the Centers for Disease Control and Prevention (CDC). Current antitoxins against BoNTs intoxication have some limitations including side effects or limited supply. As an alternative, neutralizing monoclonal antibodies will play an increasing role as BoNTs therapeutics. To date, no human anti-BoNT/B neutralizing monoclonal antibodies have yet to be reported. Herein, we describe an improved selection approach and characterization of a human monoclonal antibody, F2, which is capable of binding BoNT/B with high specificity and displays neutralizing activity in an in vitro cell-based assay. Through surface plasmon resonance studies, we have determined its association and dissociation rate constants. In sum, our data demonstrate that monoclonal antibody F2 is a promising BoNT/B therapeutic lead for further development.