New Cretaceous empidoids and the Mesozoic dance fly revolution (Diptera: Empidoidea).

Dance flies and relatives (Empidoidea) are a diverse and ecologically important group of Diptera in nearly all modern terrestrial ecosystems. Their fossil record, despite being scattered, attests to a long evolutionary history dating back to the early Mesozoic. Here, we describe seven new species of Empidoidea from Cretaceous Kachin amber inclusions, assigning them to the new genus Electrochoreutes gen.n. (type species: Electrochoreutes trisetigerus sp.n.) based on unique apomorphies among known Diptera. Like many extant dance flies, the males of Electrochoreutes are characterized by species-specific sexually dimorphic traits, which are likely to have played a role in courtship. The fine anatomy of the fossils was investigated through high-resolution X-ray phase-contrast microtomography to reconstruct their phylogenetic affinities within the empidoid clade, using cladistic reasoning. Morphology-based phylogenetic analyses including a selection of all extant family- and subfamily-ranked empidoid clades along with representatives of all extinct Mesozoic genera, were performed using a broad range of analytical methods (maximum parsimony, maximum-likelihood and Bayesian inference). These analyses converged in reconstructing Electrochoreutes as a stem-group representative of the Dolichopodidae, suggesting that complex mating rituals evolved in this lineage during the Cretaceous.

Evaluation of denoising strategies for task-based functional connectivity: Equalizing residual motion artifacts between rest and cognitively demanding tasks.

In-scanner head motion represents a major confounding factor in functional connectivity studies and it raises particular concerns when motion correlates with the effect of interest. One such instance regards research focused on functional connectivity modulations induced by sustained cognitively demanding tasks. Indeed, cognitive engagement is generally associated with substantially lower in-scanner movement compared with unconstrained, or minimally constrained, conditions. Consequently, the reliability of condition-dependent changes in functional connectivity relies on effective denoising strategies. In this study, we evaluated the ability of common denoising pipelines to minimize and balance residual motion-related artifacts between resting-state and task conditions. Denoising pipelines-including realignment/tissue-based regression, PCA/ICA-based methods (aCompCor and ICA-AROMA, respectively), global signal regression, and censoring of motion-contaminated volumes-were evaluated according to a set of benchmarks designed to assess either residual artifacts or network identifiability. We found a marked heterogeneity in pipeline performance, with many approaches showing a differential efficacy between rest and task conditions. The most effective approaches included aCompCor, optimized to increase the noise prediction power of the extracted confounding signals, and global signal regression, although both strategies performed poorly in mitigating the spurious distance-dependent association between motion and connectivity. Censoring was the only approach that substantially reduced distance-dependent artifacts, yet this came at the great cost of reduced network identifiability. The implications of these findings for best practice in denoising task-based functional connectivity data, and more generally for resting-state data, are discussed.

Investigation of the human pineal gland 3D organization by X-ray phase contrast tomography.

Pineal gland (PG) is a part of the human brain epithalamus that plays an important role in sleep, circadian rhythm, immunity, and reproduction. The calcium deposits and lesions in PG interfere with normal function of the organ and can be associated with different health disorders including serious neurological diseases. At the moment, the detailed mechanisms of PG calcifications and PG lesions formation as well as their involvement in pathological processes are not fully understood. The deep and comprehensive study of the structure of the uncut human PG with histological details, poses a stiff challenge to most imaging techniques, due to low spatial resolution, low visibility or to exceedingly aggressive sample preparation. Here, we investigate the whole uncut and unstained human post-mortem PGs by X-ray phase contrast tomography (XPCT). XPCT is an advanced 3D imaging technique, that permits to study of both soft and calcified tissue of a sample at different scales: from the whole organ to cell structure. In our research we simultaneously resolved 3D structure of parenchyma, vascular network and calcifications. Moreover, we distinguished structural details of intact and degenerated PG tissue. We discriminated calcifications with different structure, pinealocytes nuclei and the glial cells processes. All results were validated by histology. Our research clear demonstrated that XPCT is a potential tool for the high resolution 3D imaging of PG morphological features. This technique opens a new perspective to investigate PG dysfunction and understand the mechanisms of onset and progression of diseases involving the pineal gland.

X-ray phase contrast tomography for the investigation of amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. Pre-clinical studies drive the development of animal models that well mimic ALS disorder and enable both the dissection of disease processes and an early assessment of therapy efficacy. A comprehensive knowledge of neuronal and vascular lesions in the brain and spinal cord is an essential factor to understand the development of the disease. Spatial resolution and bidimensional imaging are important drawbacks limiting current neuroimaging tools, while neuropathology relies on protocols that may alter tissue chemistry and structure. In contrast, recent ex vivo studies in mice demonstrated that X-ray phase-contrast tomography enables study of the 3D distribution of both vasculature and neuronal networks, without sample sectioning or use of staining. Here we present our findings on ex vivo SOD1G93A ALS mice spinal cord at a micrometric scale. An unprecedented direct quantification of neuro-vascular alterations at different stages of the disease is shown.

Multiscale pink-beam microCT imaging at the ESRF-ID17 biomedical beamline.

Recent trends in hard X-ray micro-computed tomography (microCT) aim at increasing both spatial and temporal resolutions. These challenges require intense photon beams. Filtered synchrotron radiation beams, also referred to as `pink beams', which are emitted by wigglers or bending magnets, meet this need, owing to their broad energy range. In this work, the new microCT station installed at the biomedical beamline ID17 of the European Synchrotron is described and an overview of the preliminary results obtained for different biomedical-imaging applications is given. This new instrument expands the capabilities of the beamline towards sub-micrometre voxel size scale and simultaneous multi-resolution imaging. The current setup allows the acquisition of tomographic datasets more than one order of magnitude faster than with a monochromatic beam configuration.

Exploring Alzheimer's disease mouse brain through X-ray phase contrast tomography: From the cell to the organ.

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aß) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aß plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases.

Scale-invariant rearrangement of resting state networks in the human brain under sustained stimulation.

Brain activity at rest is characterized by widely distributed and spatially specific patterns of synchronized low-frequency blood-oxygenation level-dependent (BOLD) fluctuations, which correspond to physiologically relevant brain networks. This network behaviour is known to persist also during task execution, yet the details underlying task-associated modulations of within- and between-network connectivity are largely unknown. In this study we exploited a multi-parametric and multi-scale approach to investigate how low-frequency fluctuations adapt to a sustained n-back working memory task. We found that the transition from the resting state to the task state involves a behaviourally relevant and scale-invariant modulation of synchronization patterns within both task-positive and default mode networks. Specifically, decreases of connectivity within networks are accompanied by increases of connectivity between networks. In spite of large and widespread changes of connectivity strength, the overall topology of brain networks is remarkably preserved. We show that these findings are strongly influenced by connectivity at rest, suggesting that the absolute change of connectivity (i.e., disregarding the baseline) may not be the most suitable metric to study dynamic modulations of functional connectivity. Our results indicate that a task can evoke scale-invariant, distributed changes of BOLD fluctuations, further confirming that low frequency BOLD oscillations show a specialized response and are tightly bound to task-evoked activation.

Single cell versus large population analysis: cell variability in elemental intracellular concentration and distribution.

The quantification of elemental concentration in cells is usually performed by analytical assays on large populations missing peculiar but important rare cells. The present article aims at comparing the elemental quantification in single cells and cell population in three different cell types using a new approach for single cells elemental analysis performed at sub-micrometer scale combining X-ray fluorescence microscopy and atomic force microscopy. The attention is focused on the light element Mg, exploiting the opportunity to compare the single cell quantification to the cell population analysis carried out by a highly Mg-selective fluorescent chemosensor. The results show that the single cell analysis reveals the same Mg differences found in large population of the different cell strains studied. However, in one of the cell strains, single cell analysis reveals two cells with an exceptionally high intracellular Mg content compared with the other cells of the same strain. The single cell analysis allows mapping Mg and other light elements in whole cells at sub-micrometer scale. A detailed intensity correlation analysis on the two cells with the highest Mg content reveals that Mg subcellular localization correlates with oxygen in a different fashion with respect the other sister cells of the same strain. Graphical abstract Single cells or large population analysis this is the question!

Where is it and how much? Mapping and quantifying elements in single cells.

The biological function of a chemical element in cells not only requires the determination of its intracellular quantity, but also the spatial distribution of its concentration. Different strategies can be employed to quantify and map the intracellular concentration of elements in single cells. The assessment of the intracellular elemental concentration, which is the relevant information, requires the measurement of cell volume. This challenging and demanding task requires combining different techniques allowing gathering of both morphological and compositional information on the same cell. Moreover, the need to analyse samples more similar to their natural state requires complex hardware equipment, and supplementary efforts in preparation protocols. Nevertheless, the response to the question: "where is it and how much?" is worth all these efforts. This review aims at providing an insight into the recent and most advanced techniques and strategies for quantifying and mapping chemical elements in single cells. We describe and discuss indirect detection techniques (label based) which make use of fluorescent dyes, and direct ones (label free), such as particle induced X-ray emission, proton backscattering spectrometry, scanning transmission ion spectrometry, nano-secondary ion mass spectrometry, X-ray fluorescence microscopy, complemented by X-ray imaging.

Scale-free structural organization of oxygen interstitials in La(2)CuO(4+y).

It is well known that the microstructures of the transition-metal oxides, including the high-transition-temperature (high-T(c)) copper oxide superconductors, are complex. This is particularly so when there are oxygen interstitials or vacancies, which influence the bulk properties. For example, the oxygen interstitials in the spacer layers separating the superconducting CuO(2) planes undergo ordering phenomena in Sr(2)O(1+y)CuO(2) (ref. 9), YBa(2)Cu(3)O(6+y) (ref. 10) and La(2)CuO(4+y) (refs 11-15) that induce enhancements in the transition temperatures with no changes in hole concentrations. It is also known that complex systems often have a scale-invariant structural organization, but hitherto none had been found in high-T(c) materials. Here we report that the ordering of oxygen interstitials in the La(2)O(2+y) spacer layers of La(2)CuO(4+y) high-T(c) superconductors is characterized by a fractal distribution up to a maximum limiting size of 400 mum. Intriguingly, these fractal distributions of dopants seem to enhance superconductivity at high temperature.

Optimum inhomogeneity of local lattice distortions in La2CuO(4+y).

Electronic functionalities in materials from silicon to transition metal oxides are, to a large extent, controlled by defects and their relative arrangement. Outstanding examples are the oxides of copper, where defect order is correlated with their high superconducting transition temperatures. The oxygen defect order can be highly inhomogeneous, even in optimal superconducting samples, which raises the question of the nature of the sample regions where the order does not exist but which nonetheless form the "glue" binding the ordered regions together. Here we use scanning X-ray microdiffraction (with a beam 300 nm in diameter) to show that for La(2)CuO(4+y), the glue regions contain incommensurate modulated local lattice distortions, whose spatial extent is most pronounced for the best superconducting samples. For an underdoped single crystal with mobile oxygen interstitials in the spacer La(2)O(2+y) layers intercalated between the CuO(2) layers, the incommensurate modulated local lattice distortions form droplets anticorrelated with the ordered oxygen interstitials, and whose spatial extent is most pronounced for the best superconducting samples. In this simplest of high temperature superconductors, there are therefore not one, but two networks of ordered defects which can be tuned to achieve optimal superconductivity. For a given stoichiometry, the highest transition temperature is obtained when both the ordered oxygen and lattice defects form fractal patterns, as opposed to appearing in isolated spots. We speculate that the relationship between material complexity and superconducting transition temperature T(c) is actually underpinned by a fundamental relation between T(c) and the distribution of ordered defect networks supported by the materials.

Quantitative chemical imaging of the intracellular spatial distribution of fundamental elements and light metals in single cells.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/µm(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.

On the impact of physiological noise in spinal cord functional MRI.

Functional magnetic resonance imaging (fMRI) techniques are widely exploited for the study of brain activation. In recent years, similar approaches have been attempted for the study of spinal cord function; however, obtaining good functional images of spinal cord still represents a technical and scientific challenge. Some of the main limiting factors can be classified under the broad category of "physiological noise," and are related to 1) the cerebrospinal fluid (CSF) flux in the subarachnoid space surrounding the spinal cord; 2) the cord motion itself; and 3) the small area of the cord, which makes it critical to have a high image resolution. In addition, the different magnetic susceptibility properties of tissues surrounding the spinal cord reduce the local homogeneity of the static magnetic field, causing image distortion, reduction of the effective resolution, and signal loss, all effects that are modulated by motion. For these reasons, a number of methods have been developed for the purpose of denoising spinal cord fMRI time series. In this work, after a short introduction on the relevant features of the spinal cord anatomy, we review the main sources of physiological noise in spinal cord fMRI and discuss the main approaches useful for its mitigation.

Body size interacts with the structure of the central nervous system: A multi-center in vivo neuroimaging study.

Clinical research emphasizes the implementation of rigorous and reproducible study designs that rely on between-group matching or controlling for sources of biological variation such as subject's sex and age. However, corrections for body size (i.e. height and weight) are mostly lacking in clinical neuroimaging designs. This study investigates the importance of body size parameters in their relationship with spinal cord (SC) and brain magnetic resonance imaging (MRI) metrics. Data were derived from a cosmopolitan population of 267 healthy human adults (age 30.1±6.6 years old, 125 females). We show that body height correlated strongly or moderately with brain gray matter (GM) volume, cortical GM volume, total cerebellar volume, brainstem volume, and cross-sectional area (CSA) of cervical SC white matter (CSA-WM; 0.44≤r≤0.62). In comparison, age correlated weakly with cortical GM volume, precentral GM volume, and cortical thickness (-0.21≥r≥-0.27). Body weight correlated weakly with magnetization transfer ratio in the SC WM, dorsal columns, and lateral corticospinal tracts (-0.20≥r≥-0.23). Body weight further correlated weakly with the mean diffusivity derived from diffusion tensor imaging (DTI) in SC WM (r=-0.20) and dorsal columns (-0.21), but only in males. CSA-WM correlated strongly or moderately with brain volumes (0.39≤r≤0.64), and weakly with precentral gyrus thickness and DTI-based fractional anisotropy in SC dorsal columns and SC lateral corticospinal tracts (-0.22≥r≥-0.25). Linear mixture of sex and age explained 26±10% of data variance in brain volumetry and SC CSA. The amount of explained variance increased at 33±11% when body height was added into the mixture model. Age itself explained only 2±2% of such variance. In conclusion, body size is a significant biological variable. Along with sex and age, body size should therefore be included as a mandatory variable in the design of clinical neuroimaging studies examining SC and brain structure.

Human Pineal Gland Involutionary Process: New Findings.

In this work, we report preliminary results about the involution of the human pineal gland involution. The detailed analysis of pineal structure was done on autopsy material of 77 persons in age 27-96 using x-ray phase-contrast tomography, histology, and immunohistochemistry. Our study suggests that the pineal gland alteration in older adults may be more profound than has been reported to date. We identified and described a new form of pineal gland involution that eventually led to the total degradation of the pineal gland. To our knowledge, this study is the first to report on the complete replacement of pineal gland parenchyma with connective tissue in older adults.

Lesion Extension and Neuronal Loss after Spinal Cord Injury Using X-Ray Phase-Contrast Tomography in Mice.

Following spinal cord injury (SCI) the degree of functional (motor, autonomous, or sensory) loss correlates with the severity of nervous tissue damage. An imaging technique able to capture non-invasively and simultaneously the complex mechanisms of neuronal loss, vascular damage, and peri-lesional tissue reorganization is currently lacking in experimental SCI studies. Synchrotron X-ray phase-contrast tomography (SXPCT) has emerged as a non-destructive three-dimensional (3D) neuroimaging technique with high contrast and spatial resolution. In this framework, we developed a multi-modal approach combining SXPCT, histology and correlative methods to study neurovascular architecture in normal and spinal level C4-contused mouse spinal cords (C57BL/6J mice, age 2-3 months). The evolution of SCI lesion was imaged at the cell resolution level during the acute (30 min) and subacute (7 day) phases. Spared motor neurons (MNs) were segmented and quantified in different volumes localized at and away from the epicenter. SXPCT was able to capture neuronal loss and blood-brain barrier breakdown following SCI. Three-dimensional quantification based on SXPCT acquisitions showed no additional MN loss between 30 min and 7 days post-SCI. In addition, the analysis of hemorrhagic (at 30 min) and lesion (at 7 days) volumes revealed a high similarity in size, suggesting no extension of tissue degeneration between early and later time-points. Moreover, glial scar borders were unevenly distributed, with rostral edges being the most extended. In conclusion, SXPCT capability to image at high resolution cellular changes in 3D enables the understanding of the relationship between hemorrhagic events and nervous structure damage in SCI.

The Study of the Caudal Vertebrae of Thick-Toed Geckos after a Prolonged Space Flight by X-ray Phase-Contrast Micro-CT.

The proximal caudal vertebrae and notochord in thick-toed geckos (TG) (Chondrodactylus turneri, Gray, 1864) were investigated after a 30-day space flight onboard the biosatellite Bion-M1. This region has not been explored in previous studies. Our research focused on finding sites most affected by demineralization caused by microgravity (G0). We used X-ray phase-contrast tomography to study TG samples without invasive prior preparation to clarify our previous findings on the resistance of TG's bones to demineralization in G0. The results of the present study confirmed that geckos are capable of preserving bone mass after flight, as neither cortical nor trabecular bone volume fraction showed statistically significant changes after flight. On the other hand, we observed a clear decrease in the mineralization of the notochordal septum and a substantial rise in intercentrum volume following the flight. To monitor TG's mineral metabolism in G0, we propose to measure the volume of mineralized tissue in the notochordal septum. This technique holds promise as a sensitive approach to track the demineralization process in G0, given that the volume of calcification within the septum is limited, making it easy to detect even slight changes in mineral content.

Micromorphology of pineal gland calcification in age-related neurodegenerative diseases.

BACKGROUND: The formation of concrements in human pineal gland (PG) is a physiological process and, according to many researchers, is associated with the involution of PG structures. The majority of scientific publications concern progressive calcification of PG, leaving out studies on the destruction of already formed calcified concrements. Our study fills the gap in knowledge about calcified zones destruction in PG in normal aging and neuropathological conditions, which has not been addressed until now. PURPOSE: Our objective is to gain insight into human PG tissue impairment in both normal aging and neurodegenerative conditions. X-ray phase-contrast tomography (XPCT) allowed us to study PG tissue degeneration at high spatial resolution and, for the first time, to examine the damaged PG concrements in detail. Our research finding could potentially enhance the understanding of the PG involvement in the process of aging as well as in Alzheimer's disease (AD) and vascular dementia (VD). METHODS: The research was carried out on human PG autopsy material in normal aging, VD, and AD conditions. Laboratory-based micro-computed tomography (micro-CT) was used to collect and evaluate samples of native, uncut, and unstained PG with different degrees of pineal calcification. The detailed high-resolution 3D images of the selected PGs were produced using synchrotron-based XPCT. Histology and immunohistochemistry of soft PG tissue confirmed XPCT results. RESULTS: We performed via micro-CT the evaluation of the morphometric parameters of PG such as total sample volume, calcified concrements volume, and percentage of concrements in the total volume of the sample. XPCT imaging revealed high-resolution details of age-related PG alteration. In particular, we noted signs of moderate degradation of concrements in some PGs from elderly donors. In addition, our analysis revealed noticeable degenerative change in both concrements and soft tissue of PGs with neuropathology. In particular, we observed a hollow core and separated layers as well as deep ragged cracks in PG concrements of AD and VD samples. In parenchyma of some samples, we detected wide pinealocyte-free fluid-filled areas adjacent to the calcified zones. CONCLUSION: The present work provides the basis for future scientific research focused on the dynamic nature of PG calcium deposits and PG soft tissue in normal aging and neurodegenerative diseases.

Severity of Hepatocyte Damage and Prognosis in Cirrhotic Patients Correlate with Hepatocyte Magnesium Depletion.

We aimed to evaluate the magnesium content in human cirrhotic liver and its correlation with serum AST levels, expression of hepatocellular injury, and MELDNa prognostic score. In liver biopsies obtained at liver transplantation, we measured the magnesium content in liver tissue in 27 cirrhotic patients (CIRs) and 16 deceased donors with healthy liver (CTRLs) by atomic absorption spectrometry and within hepatocytes of 15 CIRs using synchrotron-based X-ray fluorescence microscopy. In 31 CIRs and 10 CTRLs, we evaluated the immunohistochemical expression in hepatocytes of the transient receptor potential melastatin 7 (TRPM7), a magnesium influx chanzyme also involved in inflammation. CIRs showed a lower hepatic magnesium content (117.2 (IQR 110.5-132.9) vs. 162.8 (IQR 155.9-169.8) µg/g; p < 0.001) and a higher percentage of TRPM7 positive hepatocytes (53.0 (IQR 36.8-62.0) vs. 20.7 (10.7-32.8)%; p < 0.001) than CTRLs. In CIRs, MELDNa and serum AST at transplant correlated: (a) inversely with the magnesium content both in liver tissue and hepatocytes; and (b) directly with the percentage of hepatocytes stained intensely for TRPM7. The latter also directly correlated with the worsening of MELDNa at transplant compared to waitlisting. Magnesium depletion and overexpression of its influx chanzyme TRPM7 in hepatocytes are associated with severity of hepatocyte injury and prognosis in cirrhosis. These data represent the pathophysiological basis for a possible beneficial effect of magnesium supplementation in cirrhotic patients.

Evolution and control of oxygen order in a cuprate superconductor.

The disposition of defects in metal oxides is a key attribute exploited for applications from fuel cells and catalysts to superconducting devices and memristors. The most typical defects are mobile excess oxygens and oxygen vacancies, which can be manipulated by a variety of thermal protocols as well as optical and d.c. electric fields. Here we report the X-ray writing of high-quality superconducting regions, derived from defect ordering, in the superoxygenated layered cuprate, La2CuO(4+y). Irradiation of a poor superconductor prepared by rapid thermal quenching results first in the growth of ordered regions, with an enhancement of superconductivity becoming visible only after a waiting time, as is characteristic of other systems such as ferroelectrics, where strain must be accommodated for order to become extended. However, in La2CuO(4+y), we are able to resolve all aspects of the growth of (oxygen) intercalant order, including an extraordinary excursion from low to high and back to low anisotropy of the ordered regions. We can also clearly associate the onset of high-quality superconductivity with defect ordering in two dimensions. Additional experiments with small beams demonstrate a photoresist-free, single-step strategy for writing functional materials.