Single cell imaging of nuclear architecture changes

This is the final version. Available from Frontiers Media via the DOI in this record.Data and materials availability: Data obtained in this work are available upon request.The dynamic architecture of chromatin, the macromolecular complex comprised primarily of DNA and histones, is vital for eukaryotic cell growth. Chemical and conformational changes to chromatin are important markers of functional and developmental processes in cells. However, chromatin architecture regulation has not yet been fully elucidated. Therefore, novel approaches to assessing chromatin changes at the single-cell level are required. Here we report the use of FTIR imaging and microfluidic cell-stretcher chips to assess changes to chromatin architecture and its effect on the mechanical properties of the nucleus in immune cells. FTIR imaging enables label-free chemical imaging with subcellular resolution. By optimizing the FTIR methodology and couple it with cell segmentation analysis approach, we have identified key spectral changes corresponding to changes in DNA levels and chromatin conformation at the single cell level. By further manipulating live single cells using pressure-driven microfluidics, we found that chromatin decondensation – either during general transcriptional activation or during specific immune cell maturation – can ultimately lead to nuclear auxeticity which is a new biological phenomenon recently identified. Taken together our findings demonstrate the tight and, potentially bilateral, link between extra-cellular mechanotransduction and intra-cellular nuclear architecture.Engineering and Physical Sciences Research Council (EPSRC)Biotechnology and Biological Sciences Research Council (BBSRC)Academy of Medical SciencesRoyal Societ

Laboratory activity for a new procedure of MIVIS calibration and relative validation with test data

Remotely sensed data, recorded by means of the MIVIS hyperspectral scanner in the framework of the research activity of the CNR Institutes IIA-LARA and IMAA, have been calibrated to reflectance values and then quantitatively compared with ground data. A new procedure for radiometric calibration has been defined by utilizing the MIVIS test-bench and applying a wider radiance range with respect to the one provided by the manufacturing company. New calibration curves have been determined and applied in the pre-processing chain. For validation purpose ground spectra were measured during the campaign by means of a portable spectroradiometer. The atmospheric correction has been carried out by implementing an IDL procedure to manage MODTRAN4 input and output cards. MIVIS test data acquired over Passo Corese (Roma) have shown how the new calibration coefficients significantly improve the radiometric accuracy. In particular, in the VIS spectral region the percentage error, with respect to a ground truth spectrum, is about half of that occurring if the standard calibration coefficients are used

Paleopathological study of Mammuthus meridionalis of Madonna della Strada (L’Aquila)

A skeleton of a male, 50-55 years old Mammuthus meridionalis, dated to the Pleistocene and conserved at the Spanish Fort in L’Aquila (Italy), showed a broken left tusk, in association with the presence of a deep (15x20 cm) bone erosion, involving the dental alveolus and the premaxillary bone, in close proximity to the maxillary sinus and the nasal cavity. During gross examination, small samples from three representative areas of the eroded bone were obtained. Thin sections were made and the specimens were examined under plane and polarized light, using a high resolution microscope with an incorporated digital camera. Microscopical study revealed the intra vitam origin of the lesions, characterized by the presence of woven bone fibers, typical of the early phases of bone remodeling, and lamellar bone with dilated and remodeled Haversian systems. The gross and histological findings were consistent with an osteomyelitis with bone sequestration, caused by a localized blunt trauma or, more likely, resulting from an ascending, post-traumatic chronic pulpitis, due to the tusk fracture occurred during an accident or interspecies fights. The histological exam excluded the involvement of granulomatous inflammation (e.g. tuberculosis) or neoplasia. A disease process of at least several months in duration may be hypothesized, as suggested by the histologically visible bone remodeling. A long survival of the animal after tusk loss may also be supposed, since alteration of masticatory function with altered molar teeth consumption and postural changes (i.e. atlantoaxial fusion), resulting from asymmetric weight distribution, were observed. In this study, the application of (paleo)histological techniques proved to be fundamental in order to establish the nature of bone lesions detected on archeological samples, also providing a good case for studying skull trauma and shedding light on the life history of these large mammals

Reply to comment by C. Morhange, C. Flaux, P.A. Pirazzoli, M.B. Carre on \u201cHolocene Sea level Change in Malta\u201d

The pits of Birzebbugia are located near the present-day mean sea level, and some are partially submerged. They were dated using pottery discovered in an archaeological site close to the coast, dated to the Bronze Age (Zammit, 1928; Abela, 1999). As they have been interpreted as sites for the retting of flax, during their utilization they should have remained dry and the sea could not submerge them. This is the reason why these structures are not directly related to the sea level, as suggested by Biolchi et al. (2011), so they represent an upper limit.peer-reviewe

Site-specific integration mediated by a hybrid adenovirus/adeno-associated virus vector

Adenovirus (Ad) and adeno-associated virus(AAV) have attractive and complementary properties that canbe exploited for gene transfer purposes. Ad vectors are probablythe most efficient vehicles to deliver foreign genes both invitro and in vivo. AAV exhibits the unique ability to establishlatency by efficiently integrating at a specific locus of humanchromosome 19 (AAVS1). Two viral elements are necessaryfor the integration at AAVS1: Rep68y78 and the invertedterminal repeats (AAV-ITRs). In this study, we report thedevelopment of two helper-dependent adenoviral (HD) vectors,one carrying the Rep78 gene, the other an AAV-ITRflankedtransgene. Although Rep proteins have been demonstratedto interfere with Ad replication, HD Rep78 vector wassuccessfully amplified on serial passages in 293CRE4 cellswith a yield of 50–100 transducing units per cell. DNAintegration at the AAVS1 site also was demonstrated inhepatoma cells coinfected with the HD-expressing Rep78 andwith the second HD vector carrying a transgene flanked byAAV-ITRs. The high transduction efficiency, large cloningcapacity, and high titer of the HD, combined with the sitespecificintegration machinery provided by AAV-derived components,make the AdyAAV hybrid viruses a promising vehiclefor gene therap

Single cell label-free probing of chromatin dynamics during B lymphocyte maturation

This is the final version. Available on open access from Frontiers Media via the DOI in this recordData availability: The original contributions presented in the study are publicly available. This data can be found here: https://doi.org/10.6084/m9.figshare.14135219.v1Large-scale intracellular signalling during developmental growth or in response to environmental alterations are largely orchestrated by chromatin within the cell nuclei. Chemical and conformational modifications of the chromatin architecture are critical steps in the regulation of differential gene expression and ultimately cell fate determination. Therefore, establishing chemical properties of the nucleus could provide key markers for phenotypic characterisation of cellular processes on a scale of individual cells. Raman microscopy is a sensitive technique that is capable of probing single cell chemical composition - and sub-cellular regions - in a label-free optical manner. As such, it has great potential in both clinical and basic research. However, perceived limitations of Raman spectroscopy such as low signal intensity and the difficulty in linking alterations in vibrational signals directly with ensuing biological effects have hampered advances in the field. Here we use immune B lymphocyte development as a model to assess chromatin and transcriptional changes using confocal Raman microscopy in combination with microfluidic devices and correlative transcriptomics, thereby linking changes in chemical and structural properties to biological outcomes. Live B lymphocytes were assessed before and after maturation. Multivariate analysis was applied to distinguish cellular components within each cell. The spectral differences between non-activated and activated B lymphocytes were then identified, and their correlation with known intracellular biological changes were assessed in comparison to conventional RNA-seq analysis. Our data shows that spectral analysis provides a powerful tool to study gene activation that can complement conventional molecular biology techniques and opens the way for mapping the dynamics in the biochemical makeup of individual cells.Engineering and Physical Sciences Research Council (EPSRC)Biotechnology and Biological Sciences Research Council (BBSRC)SNFRosetrees Trust FundUZH Research Priority ProgramRGSMedical Research Council (MRC

Human peopling of Italian intramontane basins: The early Middle Pleistocene site of Pagliare di Sassa (L’Aquila, central Italy)

Multidisciplinary investigations at Pagliare di Sassa (L’Aquila, central Italy) suggest that the local succession accumulated from the late Early to the early Middle Pleistocene in a lacustrine environment. In the upper part, clastic sediments are part of an alluvial fan prograding into the lake, grading to a braided fluvial system. The pollen record confirms that a significant glacial phase occurred just before the onset of the Jaramillo inversion. These data, coupled with evidence from the nearby but earlier Madonna della Strada sequence, allow reconstruction of part of the environmental evolution of L’Aquila basin before the Jaramillo Subchron. The mammal species of Pagliare di Sassa include Stephanorhinus hundsheimensis, mostly of open environments, already present at Madonna della Strada. The faunal turnover characterizing the Early to Middle Pleistocene transition is indicated by the appearances of taxa typical of the Italian early to middle Galerian faunas, such as Praemegaceros verticornis, together with Megaloceros savini. The occurrence of Mimomys savini together with Microtus ex gr. Microtus hintonigregaloides suggests that this assemblage is earlier than the Isernia La Pineta fauna. A flint implement and a fragmentary herbivore femur with impact scars probably linked to human activity give evidence of the human peopling of intramontane basins of the Apennine chain since the early Middle Pleistocene.Published170-1782.2. Laboratorio di paleomagnetismoJCR Journalrestricte

Dynamics of sun-induced chlorophyll fluorescence and reflectance to detect stress-induced variations in canopy photosynthesis

Passive measurement of sun-induced chlorophyll fluorescence (F) represents the most promising tool to quantify changes in photosynthetic functioning on a large scale. However, the complex relationship between this signal and other photosynthesis-related processes restricts its interpretation under stress conditions. To address this issue, we conducted a field campaign by combining daily airborne and ground-based measurements of F (normalized to photosynthetically active radiation), reflectance and surface temperature and related the observed changes to stress-induced variations in photosynthesis. A lawn carpet was sprayed with different doses of the herbicide Dicuran. Canopy-level measurements of gross primary productivity indicated dosage-dependent inhibition of photosynthesis by the herbicide. Dosage-dependent changes in normalized F were also detected. After spraying, we first observed a rapid increase in normalized F and in the Photochemical Reflectance Index, possibly due to the blockage of electron transport by Dicuran and the resultant impairment of xanthophyll-mediated non-photochemical quenching. This initial increase was followed by a gradual decrease in both signals, which coincided with a decline in pigment-related reflectance indices. In parallel, we also detected a canopy temperature increase after the treatment. These results demonstrate the potential of using F coupled with relevant reflectance indices to estimate stress-induced changes in canopy photosynthesis

Fast algorithm for real-time rings reconstruction

The GAP project is dedicated to study the application of GPU in several contexts in which real-time response is important to take decisions. The definition of real-time depends on the application under study, ranging from answer time of μs up to several hours in case of very computing intensive task. During this conference we presented our work in low level triggers [1] [2] and high level triggers [3] in high energy physics experiments, and specific application for nuclear magnetic resonance (NMR) [4] [5] and cone-beam CT [6]. Apart from the study of dedicated solution to decrease the latency due to data transport and preparation, the computing algorithms play an essential role in any GPU application. In this contribution, we show an original algorithm developed for triggers application, to accelerate the ring reconstruction in RICH detector when it is not possible to have seeds for reconstruction from external trackers

Microstructural imaging of the human brain with a 'super-scanner': 10 key advantages of ultra-strong gradients for diffusion MRI

The key component of a microstructural diffusion MRI 'super-scanner' is a dedicated high-strength gradient system that enables stronger diffusion weightings per unit time compared to conventional gradient designs. This can, in turn, drastically shorten the time needed for diffusion encoding, increase the signal-to-noise ratio, and facilitate measurements at shorter diffusion times. This review, written from the perspective of the UK National Facility for In Vivo MR Imaging of Human Tissue Microstructure, an initiative to establish a shared 300 mT/m-gradient facility amongst the microstructural imaging community, describes ten advantages of ultra-strong gradients for microstructural imaging. Specifically, we will discuss how the increase of the accessible measurement space compared to a lower-gradient systems (in terms of Δ, b-value, and TE) can accelerate developments in the areas of 1) axon diameter distribution mapping; 2) microstructural parameter estimation; 3) mapping micro-vs macroscopic anisotropy features with gradient waveforms beyond a single pair of pulsed-gradients; 4) multi-contrast experiments, e.g. diffusion-relaxometry; 5) tractography and high-resolution imaging in vivo and 6) post mortem; 7) diffusion-weighted spectroscopy of metabolites other than water; 8) tumour characterisation; 9) functional diffusion MRI; and 10) quality enhancement of images acquired on lower-gradient systems. We finally discuss practical barriers in the use of ultra-strong gradients, and provide an outlook on the next generation of 'super-scanners'