NGS-based identification and tracing of microsatellite instability from minute amounts DNA using inter-Alu-PCR

Sensitive detection of microsatellite instability (MSI) in tissue or liquid biopsies using next generation sequencing (NGS) has growing prognostic and predictive applications in cancer. However, the complexities of NGS make it cumbersome as compared to established multiplex-PCR detection of MSI. We present a new approach to detect MSI using inter-Alu-PCR followed by targeted NGS, that combines the practical advantages of multiplexed-PCR with the breadth of information provided by NGS. Inter-Alu-PCR employs poly-adenine repeats of variable length present in every Alu element and provides a massively-parallel, rapid approach to capture poly-A-rich genomic fractions within short 80–150bp amplicons generated from adjacent Alu-sequences. A custom-made software analysis tool, MSI-tracer, enables Alu-associated MSI detection from tissue biopsies or MSI-tracing at low-levels in circulating-DNA. MSI-associated indels at somatic-indel frequencies of 0.05–1.5% can be detected depending on the availability of matching normal tissue and the extent of instability. Due to the high Alu copy-number in human genomes, a single inter-Alu-PCR retrieves enough information for identification of MSI-associated-indels from ∼100 pg circulating-DNA, reducing current limits by ∼2-orders of magnitude and equivalent to circulating-DNA obtained from finger-sticks. The combined practical and informational advantages of inter-Alu-PCR make it a powerful tool for identifying tissue-MSI-status or tracing MSI-associated-indels in liquid biopsies.     

Weather Effects on Mobile Social Interactions: A Case Study of Mobile Phone Users in Lisbon,Portugal

The effect of weather on social interactions has been explored through the analysis of a large mobile phone use dataset. Time spent on phone calls, numbers of connected social ties, and tie strength were used as proxies for social interactions; while weather conditions were characterized in terms of temperature, relative humidity, air pressure, and wind speed. Our results are based on the analysis of a full calendar year of data for 22,696 mobile phone users (53.2 million call logs) in Lisbon, Portugal. The results suggest that different weather parameters have correlations to the level and character of social interactions. We found that although weather did not show much influence upon people''s average call duration, the likelihood of longer calls was found to increase during periods of colder weather. During periods of weather that were generally considered to be uncomfortable (i.e., very cold/warm, very low/high air pressure, and windy), people were found to be more likely to communicate with fewer social ties. Despite this tendency, we found that people are more likely to maintain their connections with those they have strong ties with much more than those of weak ties. This study sheds new light on the influence of weather conditions on social relationships and how mobile phone data can be used to investigate the influence of environmental factors on social dynamics.     

Identification and expression analysis of kcnh2 genes in the zebrafish

Long QT syndrome is a disorder that is characterised by a prolonged QT-interval and can lead to fatal cardiac arrhythmias. Many animal models have been created to study congenital long QT syndrome. Of these, zebrafish models have involved targeting two different KCNH2 gene (long QT syndrome 2) orthologues, termed zerg-2 and zerg-3, with differing cardiac phenotypes. In order to clarify this situation, this study uses a bioinformatic approach to search the current zebrafish genome sequence (Zv7 and Zv8 builds) to investigate and locate all likely zebrafish orthologues of the human KCNH2 gene. Quantitative real-time RT-PCR was also used to determine the temporal and spatial gene expression profile of the zebrafish orthologues. The data support the conclusion that zerg-2 and zerg-3 are apparent orthologues of different human genes encoding potassium ion channels, but that their functions have switched compared to the respective human proteins.     

Ultrastructural localisation of NADPH-diaphorase in the chick thymic medulla

Nicotinamide-adenine-dinucleotide phosphate-diaphorase positive cells in the chick thymus were studied at the electron-microscopic level. The formazan, a marker for the enzyme nitric oxide synthase, labelled cystic, undifferentiated, endocrine-like and myoid cells in the medulla. Some lymphoid and reticulo-epithelial cells were also lightly labelled. The reaction product was predominantly bound to the membranes of the endoplasmic reticulum in all the cells labelled and also to the nuclear envelope and outer membrane of mitochondria. The Golgi apparatus and the plasma membrane were free of the reaction product.     

A physical map of the viral genome for infectious pancreatic necrosis virus Sp: analysis of cell-free translation products derived from viral cDNA clones.

The two segments of double-stranded RNA from infectious pancreatic necrosis virus Sp were cloned into the plasmid vector pUC8. Two sets of overlapping clones were identified by restriction enzyme and Southern blot analyses. Each of these sets was shown by Northern blot analysis to be exclusively related to either segment A or B of the genomic RNA. The entire lengths of the cloned segments were estimated to be 2.9 and 2.6 kilobases, respectively. Sequences from the two segments of viral cDNA were subcloned into the bacteriophage T7 RNA polymerase vectors pT71 and pT72. The activity of the single-stranded RNAs transcribed from these subclones in a rabbit reticulocyte lysate translation system provided information on the polarity of and the protein products coded for by each subclone. The four proteins encoded by the genome of infectious pancreatic necrosis virus were identified among the translation products of the individual cloned segments by immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. By constructing plasmids containing deletions in the sequences from either the 5' or 3' end of segment A, we were able to construct a physical map for the larger segment of double-stranded RNA. The proteins derived from these plasmids indicated that the linear gene order for viral proteins encoded in segment A is beta, gamma 2, and gamma 1.     

An aspartate residue of the Yersinia pseudotuberculosis invasin protein that is critical for integrin binding.

The Yersinia pseudotuberculosis invasin protein mediates bacterial entry into mammalian cells by binding multiple beta 1-chain integrins. Invasin binding to purified alpha 5 beta 1 integrin is inhibited by Arg-Gly-Asp (RGD)-containing peptides, although invasin contains no RGD sequence. Fifteen mutations that diminished binding and bacterial entry were isolated after mutagenesis of the entire inv gene. All of the mutations altered residues within the C-terminal 192 amino acids of invasin, previously delineated as the integrin binding domain, and 10 of the mutations fell within an 11 residue region. This small region was subjected to site-directed mutagenesis and almost half of the 35 mutations generated decreased invasin-mediated entry. D911 within this region was the most critical residue, as even a conservative glutamate substitution abolished bacterial penetration. Purified invasin derivatives altered at this residue were defective in promoting cell attachment and this defect was reflected in a 10-fold or greater increase in IC50 for integrin binding. D911 may have a function similar to that of the aspartate residue in RGD-containing sequences.     

Taxonomic Position in the Genus Brucella of the Causative Agent of Canine Abortion

The gram-negative organism causing abortion in dogs was examined in parallel with cultures representative of the Brucella species and with Bordetella bronchiseptica. The organism fits into the genus Brucella and most closely resembles B. suis on the basis of its growth characteristics. It is of rough colonial morphology and is agglutinated by antisera prepared against rough Brucella. In mouse toxicity tests, no endotoxic activity could be demonstrated. In contrast to most Brucella cultures, it does not utilize erythritol. Electron microscopy showed a cell wall structure similar to that of other gram-negative organisms. The question of whether the organism should be designated Brucella canis, as proposed by Carmichael and Bruner, or Brucella suis biotype 5 is discussed. The authors favor the designation Brucella canis because the organism lacks the lipopolysaccharide antigen associated with the smooth agglutinogen and endotoxin, and it does not utilize erythritol.