Nano-imaging of environmental dust in human lung tissue by soft and hard X-ray fluorescence microscopy

It is well recognized that a large number of pulmonary diseases are induced by the effects of inhaled particulates. Anthracosis is defined as an asymptomatic, mild form of pneumoconiosis caused by the accumulation of \u201cblack carbon\u201d in the lungs due to repeated exposure to air pollution or inhalation of smoke or coal dust particles. Since the human population is progressively exposed to an increasing number and doses of anthropogenic micro and nano particles/compounds, there is a pressing urgency to explore toxicological impact arising from these exposures and the molecular mechanisms driving the body defense or possible related diseases. The toxicity mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of synchrotron radiation-based (SR-based) nano X-ray fluorescence (XRF) imaging and soft X-ray microscopy was used to chemically characterize environmental particulates (anthracosis) in lung tissues from urban subjects with the aim of better understanding the complex nature of related lungs' deposits. High-resolution XRF analyses performed at ESRF and Elettra synchrotrons allowed discriminating single particles in the heterogeneous aggregates found in the lung tissue. The small particles have variable composition resulting from the different combinations of Ti with O, K and Si, Al and Si, or Zn and Fe with O. Interestingly, simultaneous absorption and phase contrast images showed the particulate morphology and allowed to predict the presence of very dense nanoparticles or high concentration of heavy elements

Investigation of genomic DNA methylation by ultraviolet resonant Raman spectroscopy

Cytosine plays a preeminent role in DNA methylation, an epigenetic mechanism that regulates gene expression, the misregulation of which can lead to severe diseases. Several methods are nowadays employed for assessing the global DNA methylation levels, but none of them combines simplicity, high sensitivity, and low operating costs to be translated into clinical applications. Ultraviolet (UV) resonant Raman measurements at excitation wavelengths of 272 nm, 260 nm, 250 nm, and 228 nm have been carried out on isolated deoxynucleoside triphosphates (dNTPs), on a dNTP mixture as well as on genomic DNA (gDNA) samples, commercial from salmon sperm and non-commercial from B16 murine melanoma cell line. The 228 nm excitation wavelength was identified as the most suitable energy for enhancing cytosine signals over the other DNA bases. The UV Raman measurements performed at this excitation wavelength on hyper-methylated and hypo-methylated DNA from Jurkat leukemic T-cell line have revealed significant spectral differences with respect to gDNA isolated from salmon sperm and mouse melanoma B16 cells. This demonstrates how the proper choice of the excitation wavelength, combined with optimized extraction protocols, makes UV Raman spectroscopy a suitable technique for highlighting the chemical modifications undergone by cytosine nucleotides in gDNA upon hyper- and hypo-methylation events

Views of Mental Health Professionals on Positive Changes in Service Practices and Staff-User Relationships After One Year of Covid-19 Pandemic in Italy

Abstract This study explored views of mental health services (MHS) professionals regarding positive changes in service practices and organizations, and staff-user relationships after one year of COVID19 in Italy. Professionals from a community-oriented MHS completed online the Questionnaire on MHS Transformations during the COVID-19 pandemic, a 30-item tool developed by a participatory approach and validated. Of the 184 participants, 91.8% felt it was ‘‘true/definitely true’’ that during the pandemic they had informed users on procedures to reduce contagion risks, and 82.1% stated that they had increased telephone contact with users. Sixty-nine percent of professionals reported that staff revised treatment plans according to new needs of care and 78.6% stated that they had been able to mediate between user needs and safe working procedures. Moreover, 79.4% of respondents stated that they had rediscovered the importance of gestures and habits, and 65% that they had gained strength among colleagues to face fear. Fifty-four percent of participants admitted that they had discovered unexpected personal resources in users. Overall, 59.6% of participants stated that they found some positives in the COVID-19 experience. Perceived positive changes was greater among professionals from community facilities vs. those from hospital and residential facilities. In community-oriented MHS, the pandemic offered an opportunity to change practices and rethink the meaning of relationships between people. This data may be useful in generating a more balanced understanding of COVID-19’s impact on MHS and for MHS planning in the pandemic era

Impact of Sample Preparation Methods on Single-Cell X-ray Microscopy and Light Elemental Analysis Evaluated by Combined Low Energy X-ray Fluorescence, STXM and AFM

Background: Although X-ray fluorescence microscopy is becoming a widely used technique for single-cell analysis, sample preparation for this microscopy remains one of the main challenges in obtaining optimal conditions for the measurements in the X-ray regime. The information available to researchers on sample treatment is inadequate and unclear, sometimes leading to wasted time and jeopardizing the experiment's success. Many cell fixation methods have been described, but none of them have been systematically tested and declared the most suitable for synchrotron X-ray microscopy. Methods: The HEC-1-A endometrial cells, human spermatozoa, and human embryonic kidney (HEK-293) cells were fixed with organic solvents and cross-linking methods: 70% ethanol, 3.7%, and 2% paraformaldehyde; in addition, HEK-293 cells were subjected to methanol/ C3H6O treatment and cryofixation. Fixation methods were compared by coupling low-energy X-ray fluorescence with scanning transmission X-ray microscopy and atomic force microscopy. Results: Organic solvents lead to greater dehydration of cells, which has the most significant effect on the distribution and depletion of diffusion elements. Paraformaldehyde provides robust and reproducible data. Finally, the cryofixed cells provide the best morphology and element content results. Conclusion: Although cryofixation seems to be the most appropriate method as it allows for keeping cells closer to physiological conditions, it has some technical limitations. Paraformaldehyde, when used at the average concentration of 3.7%, is also an excellent alternative for X-ray microscopy

Fluorescent RT in situ PCR detection of MRP1 mRNA in human HCV infected liver.

Chronic hepatitis C is now a major cause of chronic liver disease, cirrhosis and hepatocellular carcinoma. Approximately 20% of cases of acute viral hepatitis are due to hepatitis C. Cirrhosis develops in more than 25% of patients with chronic infection and each year hepatocellular carcinoma occurs in 1-3% of patients with cirrhosis due to HCV (Hoofnagle 1999). Patients with hepatocellular carcinoma are characterized by nonresponsiveness to chemotherapeutic agents. A cause of refractivity to treatment has been ascribed to the overexpression of the Pgp (MDR) protein (Kim et al. 1999), and the additional involvement of multidrug resistance-associated proteins (MRPs) has been also hypothesized (Minemura et al. 1999)

A follow-up on psychiatric symptoms and post-traumatic stress disorders in Tuareg refugees in Burkina Faso

Introduction: The aim of this study was to carry out a 2-year follow-up of refugees in a camp in Burkina Faso who had been interviewed previously. We also aimed to verify whether the general conditions in which they lived (e.g., protection by international organizations and the conclusion of negotiations and new hope of returning to Mali and reunification with surviving family members) would affect their mental health state. Methods: This is a cross-sectional study repeated over time on a cohort of refugees. People living in the Subgandé camp who had participated in the first survey in 2012 were identified using informational chains and approached for follow-up. Those who agreed were interviewed using the Short Screening Scale for post-traumatic stress disorder (PTSD) and the K6 scale, French versions, to measure general psychopathology and the level of impairment. Results: The second survey shows a dramatic decrease in psychopathological symptoms (positivity at K6 scale). Improvement was also conspicuous in the frequency of people with stress symptoms (positivity at Short Screening Scale for PTSD and simultaneous positivity to K6 scale). The frequency of people screened positive at the Short Screening Scale for PTSD had also decreased, but the level of improvement was not pronounced. Conclusion: Our findings confirm that when physical conditions improve, psychological symptoms can also improve. Although in the studied sample psychological factors, such as the hope of returning to their own land and thus the possibility of maintaining ethnic cohesion, may have played a role, future research carried out with a proper methodology and sufficient resources to identify protective factors is needed

Morphological and Chemical Investigation of Ovarian Structures in a Bovine Model by Contrast-Enhanced X-ray Imaging and Microscopy

An improved understanding of an ovary’s structures is highly desirable to support advances in folliculogenesis knowledge and reproductive medicine, with particular attention to fertility preservation options for prepubertal girls with malignant tumors. Although currently the golden standard for structural analysis is provided by combining histological sections, staining, and visible 2D microscopic inspection, synchrotron radiation phase-contrast microtomography is becoming a new challenge for three-dimensional studies at micrometric resolution. To this aim, the proper use of contrast agents can improve the visualization of internal structures in ovary tissues, which normally present a low radiopacity. In this study, we report a comparison of four staining protocols, based on iodine or tungsten containing agents, applied to bovine ovarian tissues fixed in Bouin’s solution. The microtomography (microCT) analyses at two synchrotron facilities under different set-ups were performed at different energies in order to maximize the image contrast. While tungsten-based agents allow large structures to be well identified, Iodine ones better highlight smaller features, especially when acquired above the K-edge energy of the specific metal. Further scans performed at lower energy where the setup was optimized for overall quality and sensitivity from phase-contrast still provided highly resolved visualization of follicular and intrafollicular structures at different maturation stages, independent of the staining protocol. The analyses were complemented by X-ray Fluorescence mapping on 2D sections, showing that the tungsten-based agent has a higher penetration in this type of tissues

Gadolinium tissue deposition in the periodontal ligament of mice with reduced renal function exposed to Gd-based contrast agents

Gadolinium deposition in tissue is linked to nephrogenic systemic fibrosis (NSF): a rare disorder occurring in patients with severe chronic kidney disease and associated with administration of Gd-based contrast agents (GBCAs) for Magnetic Resonance Imaging (MRI). It is suggested that the GBCAs prolonged permanence in blood in these patients may result in a Gd precipitation in peripheral or central organs, where it initiates a fibrotic process. In this study we investigated new sites of retention/precipitation of Gd in a mouse model of renal disease (5/6 nephrectomy) receiving two doses (closely after each other) of a linear GBCA. Two commercial GBCAs (Omniscan\uae and Magnevist\uae) were administered at doses slightly higher than those used in clinical practice (0.7 mmol/kg body weight, each). The animals were sacrificed one month after the last administration and the explanted organs (kidney, liver, femur, dorsal skin, teeth) were analysed by X-ray fluorescence (XRF) at two synchrotron facilities. The XRF analysis with a millimetre-sized beam at the SYRMEP beamline (Elettra, Italy) produced no detectable levels of Gd in the examined tissues, with the notable exception of the incisors of the nephrectomised mice. The XRF analyses at sub-micron resolution performed at ID21 (ESRF, France) allowed to clearly localize Gd in the periodontal ligaments of teeth both from Omniscan\uae and Magnevist\uae treated nephrectomised mice. The latter results were further confirmed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). The study prompts that prolonged permanence of GBCAs in blood may result in Gd retention in this particular muscular tissue, opening possibilities for diagnostic applications at this level when investigating Gd-related toxicities

Differential protein folding and chemical changes in lung tissues exposed to asbestos or particulates

Environmental and occupational inhalants may induce a large number of pulmonary diseases, with asbestos exposure being the most risky. The mechanisms are clearly related to chemical composition and physical and surface properties of materials. A combination of X-ray fluorescence (\u3bcXRF) and Fourier Transform InfraRed (\u3bcFTIR) microscopy was used to chemically characterize and compare asbestos bodies versus environmental particulates (anthracosis) in lung tissues from asbestos exposed and control patients. \u3bcXRF analyses revealed heterogeneously aggregated particles in the anthracotic structures, containing mainly Si, K, Al and Fe. Both asbestos and particulates alter lung iron homeostasis, with a more marked effect in asbestos exposure. \u3bcFTIR analyses revealed abundant proteins on asbestos bodies but not on anthracotic particles. Most importantly, the analyses demonstrated that the asbestos coating proteins contain high levels of \u3b2-sheet structures. The occurrence of conformational changes in the proteic component of the asbestos coating provides new insights into long-term asbestos effects