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.     

Where Failure Is Not an Option –Personalized Medicine in Astronauts

Drug safety and efficacy are highly variable among patients. Most patients will experience the desired drug effect, but some may suffer from adverse drug reactions or gain no benefit. Pharmacogenetic testing serves as a pre-treatment diagnostic option in situations where failure or adverse events should be avoided at all costs. One such situation is human space flight. On the international space station (ISS), a list of drugs is available to cover typical emergency settings, as well as the long-term treatment of common conditions for the use in self-medicating common ailments developing over a definite period. Here, we scrutinized the list of the 78 drugs permanently available at the ISS (year 2014) to determine the extent to which their metabolism may be affected by genetic polymorphisms, potentially requiring genotype-specific dosing or choice of an alternative drug. The purpose of this analysis was to estimate the potential benefit of pharmacogenetic diagnostics in astronauts to prevent therapy failure or side effects.     

Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells

The integrase of the phage ΦC31 recombines an attP site in the phage genome with a chromosomal attB site of its Streptomyces host. We have utilized the integrase-mediated reaction to achieve episomal and genomic deletion of a reporter gene in mammalian cells, and provide the first comparison of its efficiency with other recombinases in a new assay system. This assay demonstrated that the efficiency of ΦC31-integrase is significantly enhanced by the C-terminal, but not the N-terminal, addition of a nuclear localization signal and becomes comparable with that of the widely used Cre/loxP system. Furthermore, we found that the improved FLP recombinase, FLPe, exhibits only 10% recombination activity on chromosomal targets as compared with Cre, whereas the Anabaena derived XisA recombinase is essentially inactive in mammalian cells. These results provide the first demonstration that a nuclear localisation signal and its position within a recombinase can be important for its efficiency in mammalian cells and establish the improved ΦC31-integrase as a new tool for genome engineering.