Expression study of molecular markers involved in staminality and differentiation in the colonial ascidians Botryllus schlosseri

Ascidians are invertebrate chordates, members of the subphylum Tunicata that represents the sister group of vertebrates. They offer the opportunity to investigate and compare the behaviour of both embryonic and adult stem cells. Morphological data suggest the presence of undifferentiated haemocytes (haemoblasts) able to proliferate and give rise to terminally differentiated cells. Relevant studies were also carried out in the neural lineage, in which neural progenitor cells regenerate the brain after extirpation. In B. schlosseri, during the cyclical generation change, bud primordial cells, probably deriving from a pool of long-living stem cells, are able to give rise to the neural complex. We screened the B. schlosseri genome and transcriptome, looking for transcripts/genes showing similarity to vertebrate molecular markers of haematopoietic and neural stem cells. Four sequences, orthologous to mammalian transcripts considered markers of haematopoietic progenitor cells, were identified in B. schlosseri. They are: bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6. In situ hybridization on haemocyte monolayers and colony sections, resulted in labelling of cells in the sub-endostylar haemolymph lacunae. This results matches previously morphological data that identified the endostyle as a stem cell niche. Quantitative real time PCR (qRT-PCR) highlighted the over-expression of the considered genes in the mid-cycle phase of the blastogenetic cycle. During this phase, there is the formation of new secondary buds emerging from the primary buds. The high expression levels of bsabcg2, bscd133, bsgata1/2/3 and bsgata4/5/6 genes in the mid-cycle phase reflect the presence of undifferentiated cells involved in proliferative and differentiation events required for giving rise to the new blastogenetic generation. For the neural lineage, we identified and characterised two transcripts orthologues of vertebrate neural stem cell markers (BsSox2 and BsMsi2). We also studied the expression, during the blastogenetic cycle, of a panel of genes already known to be involved in ascidian larvae neurogenesis, i.e., orthologues of Pax2/5/8, Hox1 and Hox3. ISH with riboprobes for BsSox2, BsMsi2, BsPax2/5/8, BsHox1 and BsHox3 revealed a common labelling in the endostyle niche. The presence of bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 transcripts in the cells of the region known to be a stem cell niche, led us to conclude, not only that our probes identified undifferentiated cells but even that in B. schlosseri are probably present a single population of pluripotent stem cells that could differentiate into haematopoietic or neural cells. The qRT-PCR, showed an high expression level in the mid-cycle phase of all the putative neural markers considered. In this phase new secondary buds are produced from primary buds. Each new bud needs its own neural complex and this requires the proliferation of undifferentiated cells to originate neural gland rudiment and cerebral ganglion. Bssox2, bsmsi2, bspax2/5/8, bshox1 and bshox3 increased their expression associated with these neurogenesis events and this support their involvement in neural stem cell differentiation

Pixel Detectors for Charged Particles

Pixel Detectors, as the current technology of choice for the innermost vertex detection, have reached a stage at which large detectors have been built for the LHC experiments and a new era of developments, both for hybrid and for monolithic or semi-monolithic pixel detectors is in full swing. This is largely driven by the requirements of the upgrade programme for the superLHC and by other collider experiments which plan to use monolithic pixel detectors for the first time. A review on current pixel detector developments for particle tracking and vertexing is given, comprising hybrid pixel detectors for superLHC with its own challenges in radiation and rate, as well as on monolithic, so-called active pixel detectors, including MAPS and DEPFET pixels for RHIC and superBelle.Comment: 19 pages, 23 drawings in 14 figure

Size of HIV-1 reservoir is associated with telomere shortening and immunosenescence in early-treated European children with perinatally acquired HIV-1

INTRODUCTION: Persistence of HIV-1, causing chronic immune activation, is a key determinant of premature senescence. Early antiretroviral therapy (ART) has been associated with a reduced HIV-1 reservoir in children with perinatally acquired HIV-1 (PHIV), but its impact on the senescence process is an open question. We investigated the association between HIV-1 reservoir and biological and immune ageing profile in PHIV enrolled in the multicentre cross-sectional study CARMA (Child and Adolescent Reservoir Measurements on early suppressive ART) conducted within the EPIICAL (Early treated Perinatally HIV Infected individuals: Improving Children's Actual Life) consortium. METHODS: Between September 2017 and June 2018, CARMA enrolled 40 PHIV who started ART before 2 years of age and had undetectable viremia for at least 5 years before sampling date. Samples from 37 children with a median age of 13.8 years were available for this study. HIV-1 DNA copies on CD4 cells, relative telomere length (marker of cellular senescence) and levels of T-cell receptor rearrangement excision circle (TREC, marker of thymic output) on CD4 and CD8 cells were quantified by qPCR. Immunological profile was assessed by flow cytometry. Associations between molecular and phenotypic markers, HIV-1 reservoir and age at ART initiation were explored using a multivariable Poisson regression. RESULTS: Higher HIV-1 reservoir was associated (p<0.001) with telomere shortening (incidence rate ratio [IRR] = 0.15 [0.13-0.17]), immunosenescence (CD28- CD57+ , IRR = 1.23 [1.21-1.26]) and immunoactivation (CD38+ HLADR+ , IRR = 7.29 [6.58-8.09]) of CD4 cells. Late ART initiation (after 6 months of age) correlated with higher HIV-1 reservoir levels (552 [303-1001] vs. 89 [56-365] copies/106 CD4 cells, p = 0.003) and percentage of CD4 senescent cells (2.89 [1.95-6.31] vs. 1.02 [0.45-2.69, p = 0.047). TREC levels in CD8 cells were inversely associated with HIV-1 reservoir (IRR = 0.77 [0.76-0.79]) and were significantly lower in late treated PHIV (1128 [486-1671] vs. 2278 [1425-3314], p = 0.042). CONCLUSIONS: Later ART initiation is associated with higher HIV-1 reservoir size, which correlates with increased telomere shortening and senescence of CD4 cells. Timing of ART initiation in infancy has long-term consequences on the immune and biological ageing profile of children with perinatally acquired HIV-1

Dynamics of trimming the content of face representations for categorization in the brain

To understand visual cognition, it is imperative to determine when, how and with what information the human brain categorizes the visual input. Visual categorization consistently involves at least an early and a late stage: the occipito-temporal N170 event related potential related to stimulus encoding and the parietal P300 involved in perceptual decisions. Here we sought to understand how the brain globally transforms its representations of face categories from their early encoding to the later decision stage over the 400 ms time window encompassing the N170 and P300 brain events. We applied classification image techniques to the behavioral and electroencephalographic data of three observers who categorized seven facial expressions of emotion and report two main findings: (1) Over the 400 ms time course, processing of facial features initially spreads bilaterally across the left and right occipito-temporal regions to dynamically converge onto the centro-parietal region; (2) Concurrently, information processing gradually shifts from encoding common face features across all spatial scales (e.g. the eyes) to representing only the finer scales of the diagnostic features that are richer in useful information for behavior (e.g. the wide opened eyes in 'fear'; the detailed mouth in 'happy'). Our findings suggest that the brain refines its diagnostic representations of visual categories over the first 400 ms of processing by trimming a thorough encoding of features over the N170, to leave only the detailed information important for perceptual decisions over the P300

Monolithic Active Pixel Sensors (MAPS) in a quadruple well technology for nearly 100% fill factor and full CMOS pixels

In this paper we present a novel, quadruple well process developed in a modern 0.18mu CMOS technology called INMAPS. On top of the standard process, we have added a deep P implant that can be used to form a deep P-well and provide screening of N-wells from the P-doped epitaxial layer. This prevents the collection of radiation-induced charge by unrelated N-wells, typically ones where PMOS transistors are integrated. The design of a sensor specifically tailored to a particle physics experiment is presented, where each 50mu pixel has over 150 PMOS and NMOS transistors. The sensor has been fabricated in the INMAPS process and first experimental evidence of the effectiveness of this process on charge collection is presented, showing a significant improvement in efficiency.Comment: 15 pages, 10 figures, submitted to "Sensors

Characterization of a K+-induced conformational switch in a human telomeric DNA oligonucleotide using 2-aminopurine fluorescence

Human telomeric DNA consists of tandem repeats of the DNA sequence d(GGGTTA). Oligodeoxynucletotide telomere models such as d[A(GGGTTA)(3)GGG] (Tel22) fold in a cation-dependent manner into quadruplex structures consisting of stacked G-quartets linked by d(TTA) loops. NMR has shown that in Na(+) solutions Tel22 forms a ‘basket’ topology of four antiparallel strands; in contrast, Tel22 in K(+) solutions consists of a mixture of unknown topologies. Our previous studies on the mechanism of folding of Tel22 and similar telomere analogs utilized changes in UV absorption between 270 and 325 nm that report primarily on G-quartet formation and stacking showed that quadruplex formation occurs within milliseconds upon mixing with an appropriate cation. In the current study, we assessed the dynamics and equilibria of folding of specific loops by using Tel22 derivatives in which the dA residues were serially substituted with the fluorescent reporter base, 2-aminopurine (2-AP). Tel22 folding induced by Na(+) or K(+) assessed by changes in 2-AP fluorescence consists of at least three kinetic steps with time constants spanning a range of ms to several hundred seconds. Na(+)-dependent equilibrium titrations of Tel22 folding could be approximated as a cooperative two-state process. In contrast, K(+)-dependent folding curves were biphasic, revealing that different conformational ensembles are present in 1 mM and 30 mM K(+). This conclusion was confirmed by (1)H NMR. Molecular dynamics simulations revealed a K(+) binding pocket in Tel22 located near dA1 that is specific for the so-called hybrid-1 conformation in which strand 1 is in a parallel arrangement. The possible presence of this topologically specific binding site suggests that K(+) may play an allosteric role in regulating telomere conformation and function by modulating quadruplex tertiary structure

Rapid detection of peptide markers for authentication purposes in raw and cooked meat using ambient liquid extraction surface analysis mass spectrometry

In this paper, our previously developed ambient LESA-MS methodology is implemented to analyze five types of thermally treated meat species, namely beef, pork, horse, chicken, and turkey meat, in order to select and identify heat-stable and species-specific peptide markers. In-solution tryptic digests of cooked meats were deposited onto a polymer surface, followed by LESA-MS analysis and evaluation using multivariate data analysis and tandem electrospray MS. The five types of cooked meat were clearly discriminated using principal component analysis and orthogonal partial least squares discriminant analysis. A number of 23 heat stable peptide markers unique to species and muscle protein were identified following data-dependent tandem LESA-MS analysis. Surface extraction and direct ambient MS analysis of mixtures of cooked meat species was performed for the first time and enabled detection of 10% (w/w) of pork, horse, and turkey meat, and 5% (w/w) of chicken meat in beef, using the developed LESA-MS/MS analysis. The study shows, for the first time, that ambient LESA-MS methodology displays specificity sufficient to be implemented effectively for the analysis of processed and complex peptide digests. The proposed approach is much faster and simpler than other measurement tools for meat speciation; it has potential for application in other areas of meat science or food production

Search for New Physics with Jets and Missing Transverse Momentum in pp collisions at sqrt(s) = 7 TeV

A search for new physics is presented based on an event signature of at least three jets accompanied by large missing transverse momentum, using a data sample corresponding to an integrated luminosity of 36 inverse picobarns collected in proton--proton collisions at sqrt(s)=7 TeV with the CMS detector at the LHC. No excess of events is observed above the expected standard model backgrounds, which are all estimated from the data. Exclusion limits are presented for the constrained minimal supersymmetric extension of the standard model. Cross section limits are also presented using simplified models with new particles decaying to an undetected particle and one or two jets