CGH and SNP array using DNA extracted from fixed cytogenetic preparations and long-term refrigerated bone marrow specimens

<p>Abstract</p> <p>Background</p> <p>The analysis of nucleic acids is limited by the availability of archival specimens and the quality and amount of the extracted material. Archived cytogenetic preparations are stored in many laboratories and are a potential source of total genomic DNA for array karyotyping and other applications. Array CGH using DNA from fixed cytogenetic preparations has been described, but it is not known whether it can be used for SNP arrays. Diagnostic bone marrow specimens taken during the assessment of hematological malignancies are also a potential source of DNA, but it is generally assumed that DNA must be extracted, or the specimen frozen, within a day or two of collection, to obtain DNA suitable for further analysis. We have assessed DNA extracted from these materials for both SNP array and array CGH.</p> <p>Results</p> <p>We show that both SNP array and array CGH can be performed on genomic DNA extracted from cytogenetic specimens stored in Carnoy's fixative, and from bone marrow which has been stored unfrozen, at 4°C, for at least 36 days. We describe a procedure for extracting a usable concentration of total genomic DNA from cytogenetic suspensions of low cellularity.</p> <p>Conclusions</p> <p>The ability to use these archival specimens for DNA-based analysis increases the potential for retrospective genetic analysis of clinical specimens. Fixed cytogenetic preparations and long-term refrigerated bone marrow both provide DNA suitable for array karyotyping, and may be suitable for a wider range of analytical procedures.</p

The Meningococcal Vaccine Candidate Neisserial Surface Protein A (NspA) Binds to Factor H and Enhances Meningococcal Resistance to Complement

Complement forms an important arm of innate immunity against invasive meningococcal infections. Binding of the alternative complement pathway inhibitor factor H (fH) to fH-binding protein (fHbp) is one mechanism meningococci employ to limit complement activation on the bacterial surface. fHbp is a leading vaccine candidate against group B Neisseria meningitidis. Novel mechanisms that meningococci employ to bind fH could undermine the efficacy of fHbp-based vaccines. We observed that fHbp deletion mutants of some meningococcal strains showed residual fH binding suggesting the presence of a second receptor for fH. Ligand overlay immunoblotting using membrane fractions from one such strain showed that fH bound to a ∼17 kD protein, identified by MALDI-TOF analysis as Neisserial surface protein A (NspA), a meningococcal vaccine candidate whose function has not been defined. Deleting nspA, in the background of fHbp deletion mutants, abrogated fH binding and mAbs against NspA blocked fH binding, confirming NspA as a fH binding molecule on intact bacteria. NspA expression levels vary among strains and expression correlated with the level of fH binding; over-expressing NspA enhanced fH binding to bacteria. Progressive truncation of the heptose (Hep) I chain of lipooligosaccharide (LOS), or sialylation of lacto-N-neotetraose LOS both increased fH binding to NspA-expressing meningococci, while expression of capsule reduced fH binding to the strains tested. Similar to fHbp, binding of NspA to fH was human-specific and occurred through fH domains 6–7. Consistent with its ability to bind fH, deleting NspA increased C3 deposition and resulted in increased complement-dependent killing. Collectively, these data identify a key complement evasion mechanism with important implications for ongoing efforts to develop meningococcal vaccines that employ fHbp as one of its components

The Sloan Digital Sky Survey: Technical Summary

The Sloan Digital Sky Survey (SDSS) will provide the data to support detailed investigations of the distribution of luminous and non- luminous matter in the Universe: a photometrically and astrometrically calibrated digital imaging survey of pi steradians above about Galactic latitude 30 degrees in five broad optical bands to a depth of g' about 23 magnitudes, and a spectroscopic survey of the approximately one million brightest galaxies and 10^5 brightest quasars found in the photometric object catalog produced by the imaging survey. This paper summarizes the observational parameters and data products of the SDSS, and serves as an introduction to extensive technical on-line documentation.Comment: 9 pages, 7 figures, AAS Latex. To appear in AJ, Sept 200

The formation and fate of internal waves in the South China Sea

Author Posting. © The Author(s), 2015. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in Nature 521 (2015): 65-69, doi:10.1038/nature14399.Internal gravity waves, the subsurface analogue of the familiar surface gravity waves that break on beaches, are ubiquitous in the ocean. Because of their strong vertical and horizontal currents, and the turbulent mixing caused by their breaking, they impact a panoply of ocean processes, such as the supply of nutrients for photosynthesis1, sediment and pollutant transport2 and acoustic transmission3; they also pose hazards for manmade structures in the ocean4. Generated primarily by the wind and the tides, internal waves can travel thousands of kilometres from their sources before breaking5, posing severe challenges for their observation and their inclusion in numerical climate models, which are sensitive to their effects6-7. Over a decade of studies8-11 have targeted the South China Sea, where the oceans’ most powerful internal waves are generated in the Luzon Strait and steepen dramatically as they propagate west. Confusion has persisted regarding their generation mechanism, variability and energy budget, however, due to the lack of in-situ data from the Luzon Strait, where extreme flow conditions make measurements challenging. Here we employ new observations and numerical models to (i) show that the waves begin as sinusoidal disturbances rather than from sharp hydraulic phenomena, (ii) reveal the existence of >200-m-high breaking internal waves in the generation region that give rise to turbulence levels >10,000 times that in the open ocean, (iii) determine that the Kuroshio western boundary current significantly refracts the internal wave field emanating from the Luzon Strait, and (iv) demonstrate a factor-of-two agreement between modelled and observed energy fluxes that enables the first observationally-supported energy budget of the region. Together, these findings give a cradle-to-grave picture of internal waves on a basin scale, which will support further improvements of their representation in numerical climate predictions.Our work was supported by the U.S. Office of Naval Research and the Taiwan National Science Council.2015-10-2

The formation and fate of internal waves in the South China Sea

Internal gravity waves, the subsurface analogue of the familiar surface gravity waves that break on beaches, are ubiquitous in the ocean. Because of their strong vertical and horizontal currents, and the turbulent mixing caused by their breaking, they affect a panoply of ocean processes, such as the supply of nutrients for photosynthesis¹, sediment and pollutant transport² and acoustic transmission³; they also pose hazards for man-made structures in the ocean⁴. Generated primarily by the wind and the tides, internal waves can travel thousands of kilometres from their sources before breaking⁵, making it challenging to observe them and to include them in numerical climate models, which are sensitive to their effects[superscript 6,7]. For over a decade, studies[superscript 8–11] have targeted the South China Sea, where the oceans’ most powerful known internal waves are generated in the Luzon Strait and steepen dramatically as they propagate west. Confusion has persisted regarding their mechanism of generation, variability and energy budget, however, owing to the lack of in situ data from the Luzon Strait, where extreme flow conditions make measurements difficult. Here we use new observations and numerical models to (1) show that the waves begin as sinusoidal disturbances rather than arising from sharp hydraulic phenomena, (2) reveal the existence of >200-metre-high breaking internal waves in the region of generation that give rise to turbulence levels >10,000 times that in the open ocean, (3) determine that the Kuroshio western boundary current noticeably refracts the internal wave field emanating from the Luzon Strait, and (4) demonstrate a factor-of-two agreement between modelled and observed energy fluxes, which allows us to produce an observationally supported energy budget of the region. Together, these findings give a cradle-to-grave picture of internal waves on a basin scale, which will support further improvements of their representation in numerical climate predictions

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival