Isolation and characterization of circulating tumor cells using a novel workflow combining the CellSearch® system and the CellCelector™

Circulating tumor cells (CTC) are rare cells which have left the primary tumor to enter the blood stream. Although only a small CTC subgroup is capable of extravasating, the presence of CTCs is associated with an increased risk of metastasis and a shorter overall survival. Understanding the heterogeneous CTC biology will optimize treatment decisions and will thereby improve patient outcome. For this, robust workflows for detection and isolation of CTCs are urgently required. Here, we present a workflow to characterize CTCs by combining the advantages of both the CellSearch^® and the CellCelector? micromanipulation system. CTCs were isolated from CellSearch^® cartridges using the CellCelector? system and were deposited into PCR tubes for subsequent molecular analysis (whole genome amplification (WGA) and massive parallel multigene sequencing). By a CellCelector? screen we reidentified 97% of CellSearch^® SKBR‐3 cells. Furthermore, we isolated 97% of CellSearch^®‐proven patient CTCs using the CellCelector? system. Therein, we found an almost perfect correlation of R² = 0.98 (Spearman's rho correlation, n = 20, p < 0.00001) between the CellSearch^® CTC count (n = 271) and the CellCelector? detected CTCs (n = 252). Isolated CTCs were analyzed by WGA and massive parallel multigene sequencing. In total, single nucleotide polymorphisms (SNPs) could be detected in 50 genes in seven CTCs, 12 MCF‐7, and 3 T47D cells, respectively. Taken together, CTC quantification via the CellCelector? system ensures a comprehensive detection of CTCs preidentified by the CellSearch^® system. Moreover, the isolation of CTCs after CellSearch^® using the CellCelector? system guarantees for CTC enrichment without any contaminants enabling subsequent high throughput genomic analyses on single cell level. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 33:125–132, 2017     

A strategy for the characterization of minute chromosome rearrangements using multiple color fluorescence in situ hybridization with chromosome-specific DNA libraries and YAC clones

The identification of marker chromosomes in clinical and tumor cytogenetics by chromosome banding analysis can create problems. In this study, we present a strategy to define minute chromosomal rearrangements by multicolor fluorescence in situ hybridization (FISH) with whole chromosome painting probes derived from chromosome-specific DNA libraries and Alu-polymerase chain reaction (PCR) products of various region-specific yeast artificial chromosome (YAC) clones. To demonstrate the usefulness of this strategy for the characterization of chromosome rearrangements unidentifiable by banding techniques, an 8p+ marker chromosome with two extra bands present in the karyotype of a child with multiple anomalies, malformations, and severe mental retardation was investigated. A series of seven-color FISH experiments with sets of fluorochrome-labeled DNA library probes from flow-sorted chromosomes demonstrated that the additional segment on 8p+ was derived from chromosome 6. For a more detailed characterization of the marker chromosome, three-color FISH experiments with library probes specific to chromosomes 6 and 8 were performed in combination with newly established telomeric and subtelomeric YAC clones from 6q25, 6p23, and 8p23. These experiments demonstrated a trisomy 6pter6p22 and a monosomy 8pter8p23 in the patient. The present limitations for a broad application of this strategy and its possible improvements are discussed.Dedicated to Professor Dr. U. Wolf on the occasion of his 60th birthday     

Identification of a neuropeptide S responsive circuitry shaping amygdala activity via the endopiriform nucleus

Neuropeptide S (NPS) and its receptor are thought to define a set of specific brain circuits involved in fear and anxiety. Here we provide evidence for a novel, NPS-responsive circuit that shapes neural activity in the mouse basolateral amygdala (BLA) via the endopiriform nucleus (EPN). Using slice preparations, we demonstrate that NPS directly activates an inward current in 20% of EPN neurons and evokes an increase of glutamatergic excitation in this nucleus. Excitation of the EPN is responsible for a modulation of BLA activity through NPS, characterized by a general increase of GABAergic inhibition and enhancement of spike activity in a subset of BLA projection neurons. Finally, local injection of NPS to the EPN interferes with the expression of contextual, but not auditory cued fear memory. Together, these data suggest the existence of a specific NPS-responsive circuitry between EPN and BLA, likely involved in contextual aspects of fear memory.     

Two-dimensional gel-based alkaline proteome of the probiotic bacterium Lactobacillus acidophilus NCFM

Lactobacillus acidophilus NCFM (NCFM) is a well-documented probiotic bacterium isolated from human gut. Detailed 2D gel-based NCFM proteomics addressed the so-called alkaline range, i.e., pH 6-11. Proteins were identified in 150 of the 202 spots picked from the Coomassie Brilliant Blue stained 2D gel using MALDI-TOF-MS. The 102 unique gene products among the 150 protein identifications were assigned to different functional categories, and evaluated by considering a calculated distribution of abundance as well as grand average of hydrophobicity values. None of the very few available lactic acid bacteria proteome reference maps included the range of pI >7.0. The present report of such data on the proteome of NCFM fundamentally complements current knowledge on protein profiles limited to the acid and neutral pH range.     

Phylogenetic signals in thermal traits remain stronger in the tropics if we can believe published physiological data. A reply to McKechnie et al., “Data quality problems undermine analyses of endotherm upper critical temperatures”

Due to concerns about data quality, McKechnie, Coe, Gerson, and Wolf ( 2016 ) questioned the conclusions of our study (Khaliq et al., 2015 ) published in this journal. Here, we argue that most of the questioned data points are in fact useful for macrophysiological analyses, mostly because the vast majority of data are explicitly reported in the peer‐reviewed physiological literature. Furthermore, we show that our conclusions remain largely robust irrespective of the data inclusion criterion. While we think that constructive debates about the adequate use of primary data in meta‐studies as well as more transparency in data inclusion criteria are indeed useful, we also emphasize that data suitability should be evaluated in the light of the scope and scale of the study in which they are used. We hope that this discussion will not discourage the exchange between disciplines such as biogeography and physiology, as this integration is needed to address some of the most urgent scientific challenges.     

Telomere Biology and Cellular Aging in Nonhuman Primate Cells

To determine how cellular aging is conserved among primates, we analyzed the replicative potential and telomere shortening in skin fibroblasts of anthropoids and prosimians. The average telomere length of the New World primates Ateles geoffroyi (spider monkey) and Saimiri sciureus (squirrel monkey) and the Old World primates Macaca mulatta (rhesus monkey), Pongo pygmaeus (orangutan), and Pan paniscus (pigmy chimpanzee) ranged from 4 to 16 kb. We found that telomere shortening limits the replicative capacity of anthropoid fibroblasts and that the expression of human telomerase produced telomere elongation and the extension of their in vitro life span. In contrast the prosimian Lemur catta (ring-tailed lemur) had both long and short telomeres and telomere shortening did not provide an absolute barrier to immortalization. Following a transient growth arrest a subset of cells showing a reduced number of chromosomes overgrew the cultures without activation of telomerase. Here we show that the presence of continuous TTAGGG repeats at telomeres and rigorous control of replicative aging by telomere shortening appear to be conserved among anthropoid primates but is less effective in prosimian lemurs.     

Available data point to a 4‐km‐high Tibetan Plateau by 40 Ma,but 100 molecular‐clock papers have linked supposed recent uplift to young node ages

The aims of this study were to synthesize data on the orogeny of the Tibetan Plateau (TP), with a focus on its elevation since the collision of the Eurasian and Indian plates, and to review the arguments in 100 phylogeny‐cum‐biogeography papers that have linked young inferred divergence times to recent TP uplift phases. I surveyed the literature on the geological history of the TP, focusing on different types of data used to infer its past height. I also tabulated the supposed TP history (and supporting references) in papers since 1998. Since the early 1990s, evidence from tectonics, isotopes, fossils and climate simulations increasingly indicates that the TP has been 4–5 km high since the mid‐Eocene. The data also indicate that the Indian summer monsoon, South‐east Asian summer monsoon, and Central Asian winter monsoon arose at different times and are unrelated to Tibetan uplift. A growing number of studies by biologists, however, are linking node ages between 0.5 and 15 Ma to specific (author‐dependent) uplift phases of the TP citing geological papers that are outdated or miscited. Biogeography of the TP thus currently appears to be in a self‐created bubble that encloses hundreds of authors and referees. Our understanding of the biogeography of Tibet requires up‐to‐date interpretation of its geological history and more fieldwork on local ecological habitat diversity, the plateau's history during the Pleistocene and the distribution of possible refugia.