A total transcriptome profiling method for plasma-derived extracellular vesicles: applications for liquid biopsies

Extracellular vesicles (EVs) are key mediators of intercellular communication. Part of their biological effects can be attributed to the transfer of cargos of diverse types of RNAs, which are promising diagnostic and prognostic biomarkers. EVs found in human biofluids are a valuable source for the development of minimally invasive assays. However, the total transcriptional landscape of EVs is still largely unknown. Here we develop a new method for total transcriptome profiling of plasma-derived EVs by next generation sequencing (NGS) from limited quantities of patient-derived clinical samples, which enables the unbiased characterization of the complete RNA cargo, including both small- and long-RNAs, in a single library preparation step. This approach was applied to RNA extracted from EVs isolated by ultracentrifugation from the plasma of five healthy volunteers. Among the most abundant RNAs identified we found small RNAs such as tRNAs, miRNAs and miscellaneous RNAs, which have largely unknown functions. We also identified protein-coding and long noncoding transcripts, as well as circular RNA species that were also experimentally validated. This method enables, for the first time, the full spectrum of transcriptome data to be obtained from minute patient-derived samples, and will therefore potentially allow the identification of cell-to-cell communication mechanisms and biomarkers.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESP)Gillson-Longenbaugh FoundationNational Institutes of Health (NIH/NCATS) through the NIH Common Fund, Office of Strategic Coordination (OSC)AC Camargo Canc Ctr, Lab Med Genom, Sao Paulo, SP, BrazilAC Camargo Canc Ctr, Lab Computat Biol, Sao Paulo, SP, BrazilUniv Sao Paulo, Inst Biomed Sci, Dept Cell & Dev Biol, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Electron Microscopy Ctr, Sao Paulo, SP, BrazilUniv Texas MD Anderson Canc Ctr, Dept Expt Therapeut, Houston, TX 77030 USAUniv Texas MD Anderson Canc Ctr, Ctr RNA Interference & Non Coding RNAs, Houston, TX 77030 USAUniv New Mexico, Comprehens Canc Ctr, Albuquerque, NM 87131 USAUniv New Mexico, Sch Med, Div Hematol Oncol, Dept Internal Med, Albuquerque, NM 87131 USAUniv New Mexico, Sch Med, Div Mol Med, Dept Internal Med, Albuquerque, NM 87131 USARockefeller Univ, Lab Mol Immunol, 1230 York Ave, New York, NY 10021 USAFMUSP, Lab Neurociencias Alzira Denise Hertzog Silva LIM, Inst Psiquiatria, Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Electron Microscopy Ctr, Sao Paulo, SP, BrazilFAPESP: 2011/09172-3FAPESP: 2014/26897-0Web of Scienc

Massive amplitudes on the Coulomb branch of N=4 SYM

We initiate a systematic study of amplitudes with massive external particles on the Coulomb-branch of N=4 super Yang Mills theory: 1) We propose that (multi-)soft-scalar limits of massless amplitudes at the origin of moduli space can be used to determine Coulomb-branch amplitudes to leading order in the mass. This is demonstrated in numerous examples. 2) We find compact explicit expressions for several towers of tree-level amplitudes, including scattering of two massive W-bosons with any number of positive helicity gluons, valid for all values of the mass. 3) We present the general structure of superamplitudes on the Coulomb branch. For example, the n-point "MHV-band" superamplitude is proportional to a Grassmann polynomial of mixed degree 4 to 12, which is uniquely determined by supersymmetry. We find explicit tree-level superamplitudes for this MHV band and for other simple sectors of the theory. 4) Dual conformal generators are constructed, and we explore the dual conformal properties of the simplest massive amplitudes. Our compact expressions for amplitudes and superamplitudes should be of both theoretical and phenomenological interest; in particular the tree-level results carry over to truncations of the theory with less supersymmetry.Comment: 29 pages, 1 figur

Detection of oxaliplatin- and cisplatin-DNA lesions requires different global genome repair mechanisms that affect their clinical efficacy

The therapeutic efficacy of cisplatin and oxaliplatin depends on the balance between the DNA damage induction and the DNA damage response of tumor cells. Based on clinical evidence, oxaliplatin is administered to cisplatin-unresponsive cancers, but the underlying molecular causes for this tumor specificity are not clear. Hence, stratification of patients based on DNA repair profiling is not sufficiently utilized for treatment selection. Using a combination of genetic, transcriptomics and imaging approaches, we identified factors that promote global genome nucleotide excision repair (GG-NER) of DNA-platinum adducts induced by oxaliplatin, but not by cisplatin. We show that oxaliplatin-DNA lesions are a poor substrate for GG-NER initiating factor XPC and that DDB2 and HMGA2 are required for efficient binding of XPC to oxaliplatin lesions and subsequent GG-NER initiation. Loss of DDB2 and HMGA2 therefore leads to hypersensitivity to oxaliplatin but not to cisplatin. As a result, low DDB2 levels in different colon cancer cells are associated with GG-NER deficiency and oxaliplatin hypersensitivity. Finally, we show that colon cancer patients with low DDB2 levels have a better prognosis after oxaliplatin treatment than patients with high DDB2 expression. We therefore propose that DDB2 is a promising predictive marker of oxaliplatin treatment efficiency in colon cancer.</p

DUVET Survey: Mapping Outflows in the Metal-Poor Starburst Mrk 1486

We present a method to characterize star-formation driven outflows from edge-on galaxies and apply this method to the metal-poor starburst galaxy, Mrk 1486. Our method uses the distribution of emission line flux (from H$\beta$ and [OIII] 5007) to identify the location of the outflow and measure the extent above the disk, the opening angle, and the transverse kinematics. We show that this simple technique recovers a similar distribution of the outflow without requiring complex modelling of line-splitting or multi-Gaussian components, and is therefore applicable to lower spectral resolution data. In Mrk 1486 we observe an asymmetric outflow in both the location of the peak flux and total flux from each lobe. We estimate an opening angle of $17-37^{\circ}$ depending on the method and assumptions adopted. Within the minor axis outflows, we estimate a total mass outflow rate of $\sim2.5$ M$_{\odot}$ yr$^{-1}$, which corresponds to a mass loading factor of $\eta=0.7$. We observe a non-negligible amount of flux from ionized gas outflowing along the edge of the disk (perpendicular to the biconical components), with a mass outflow rate $\sim0.9$ M$_{\odot}$ yr$^{-1}$. Our results are intended to demonstrate a method that can be applied to high-throughput, low spectral resolution observations, such as narrow band filters or low spectral resolution IFS that may be more able to recover the faint emission from outflows.Comment: 12 Pages, 6 Figure

Shape modeling technique KOALA validated by ESA Rosetta at (21) Lutetia

We present a comparison of our results from ground-based observations of asteroid (21) Lutetia with imaging data acquired during the flyby of the asteroid by the ESA Rosetta mission. This flyby provided a unique opportunity to evaluate and calibrate our method of determination of size, 3-D shape, and spin of an asteroid from ground-based observations. We present our 3-D shape-modeling technique KOALA which is based on multi-dataset inversion. We compare the results we obtained with KOALA, prior to the flyby, on asteroid (21) Lutetia with the high-spatial resolution images of the asteroid taken with the OSIRIS camera on-board the ESA Rosetta spacecraft, during its encounter with Lutetia. The spin axis determined with KOALA was found to be accurate to within two degrees, while the KOALA diameter determinations were within 2% of the Rosetta-derived values. The 3-D shape of the KOALA model is also confirmed by the spectacular visual agreement between both 3-D shape models (KOALA pre- and OSIRIS post-flyby). We found a typical deviation of only 2 km at local scales between the profiles from KOALA predictions and OSIRIS images, resulting in a volume uncertainty provided by KOALA better than 10%. Radiometric techniques for the interpretation of thermal infrared data also benefit greatly from the KOALA shape model: the absolute size and geometric albedo can be derived with high accuracy, and thermal properties, for example the thermal inertia, can be determined unambiguously. We consider this to be a validation of the KOALA method. Because space exploration will remain limited to only a few objects, KOALA stands as a powerful technique to study a much larger set of small bodies using Earth-based observations.Comment: 15 pages, 8 figures, 2 tables, accepted for publication in P&S

DUVET: Spatially Resolved Observations of Star Formation Regulation via Galactic Outflows in a Starbursting Disk Galaxy

We compare 500~pc scale, resolved observations of ionised and molecular gas for the $z\sim0.02$ starbursting disk galaxy IRAS08339+6517, using measurements from KCWI and NOEMA. We explore the relationship of the star formation driven ionised gas outflows with colocated galaxy properties. We find a roughly linear relationship between the outflow mass flux ($\dot{\Sigma}_{\rm out}$) and star formation rate surface density ($\Sigma_{\rm SFR}$), $\dot{\Sigma}_{\rm out}\propto\Sigma_{\rm SFR}^{1.06\pm0.10}$, and a strong correlation between $\dot{\Sigma}_{\rm out}$ and the gas depletion time, such that $\dot{\Sigma}_{\rm out} \propto t_{dep}^{-1.1\pm0.06}$. Moreover, we find these outflows are so-called ``breakout" outflows, according to the relationship between the gas fraction and disk kinematics. Assuming that ionised outflow mass scales with total outflow mass, our observations suggest that the regions of highest $\Sigma_{\rm SFR}$ in IRAS08 are removing more gas via the outflow than through the conversion of gas into stars. Our results are consistent with a picture in which the outflow limits the ability for a region of a disk to maintain short depletion times. Our results underline the need for resolved observations of outflows in more galaxies.Comment: 16 pages, 7 figures, Submitted to Ap

NOMAD spectrometer on the ExoMars trace gas orbiter mission: part 2—design, manufacturing, and testing of the ultraviolet and visible channel

NOMAD is a spectrometer suite on board the ESA/Roscosmos ExoMars Trace Gas Orbiter, which launched in March 2016. NOMAD consists of two infrared channels and one ultraviolet and visible channel, allowing the instrument to perform observations quasi-constantly, by taking nadir measurements at the day- and night-side, and during solar occultations. Here, in part 2 of a linked study, we describe the design, manufacturing, and testing of the ultraviolet and visible spectrometer channel called UVIS. We focus upon the optical design and working principle where two telescopes are coupled to a single grating spectrometer using a selector mechanism

Ultrafast Evolution and Loss of CRISPRs Following a Host Shift in a Novel Wildlife Pathogen, Mycoplasma gallisepticum

Measureable rates of genome evolution are well documented in human pathogens but are less well understood in bacterial pathogens in the wild, particularly during and after host switches. Mycoplasma gallisepticum (MG) is a pathogenic bacterium that has evolved predominantly in poultry and recently jumped to wild house finches (Carpodacus mexicanus), a common North American songbird. For the first time we characterize the genome and measure rates of genome evolution in House Finch isolates of MG, as well as in poultry outgroups. Using whole-genome sequences of 12 House Finch isolates across a 13-year serial sample and an additional four newly sequenced poultry strains, we estimate a nucleotide diversity in House Finch isolates of only ∼2% of ancestral poultry strains and a nucleotide substitution rate of 0.8−1.2×10−5 per site per year both in poultry and in House Finches, an exceptionally fast rate rivaling some of the highest estimates reported thus far for bacteria. We also found high diversity and complete turnover of CRISPR arrays in poultry MG strains prior to the switch to the House Finch host, but after the invasion of House Finches there is progressive loss of CRISPR repeat diversity, and recruitment of novel CRISPR repeats ceases. Recent (2007) House Finch MG strains retain only ∼50% of the CRISPR repertoire founding (1994–95) strains and have lost the CRISPR–associated genes required for CRISPR function. Our results suggest that genome evolution in bacterial pathogens of wild birds can be extremely rapid and in this case is accompanied by apparent functional loss of CRISPRs

Using Classical Population Genetics Tools with Heterochroneous Data: Time Matters!

BACKGROUND:New polymorphism datasets from heterochroneous data have arisen thanks to recent advances in experimental and microbial molecular evolution, and the sequencing of ancient DNA (aDNA). However, classical tools for population genetics analyses do not take into account heterochrony between subsets, despite potential bias on neutrality and population structure tests. Here, we characterize the extent of such possible biases using serial coalescent simulations. METHODOLOGY/PRINCIPAL FINDINGS:We first use a coalescent framework to generate datasets assuming no or different levels of heterochrony and contrast most classical population genetic statistics. We show that even weak levels of heterochrony ( approximately 10% of the average depth of a standard population tree) affect the distribution of polymorphism substantially, leading to overestimate the level of polymorphism theta, to star like trees, with an excess of rare mutations and a deficit of linkage disequilibrium, which are the hallmark of e.g. population expansion (possibly after a drastic bottleneck). Substantial departures of the tests are detected in the opposite direction for more heterochroneous and equilibrated datasets, with balanced trees mimicking in particular population contraction, balancing selection, and population differentiation. We therefore introduce simple corrections to classical estimators of polymorphism and of the genetic distance between populations, in order to remove heterochrony-driven bias. Finally, we show that these effects do occur on real aDNA datasets, taking advantage of the currently available sequence data for Cave Bears (Ursus spelaeus), for which large mtDNA haplotypes have been reported over a substantial time period (22-130 thousand years ago (KYA)). CONCLUSIONS/SIGNIFICANCE:Considering serial sampling changed the conclusion of several tests, indicating that neglecting heterochrony could provide significant support for false past history of populations and inappropriate conservation decisions. We therefore argue for systematically considering heterochroneous models when analyzing heterochroneous samples covering a large time scale