Cryopreservation affects platelet macromolecular composition over time after thawing and differently impacts on cancer cells behavior in vitro.

Cryopreservation affects platelets' function, questioning their use for cancer patients. We aimed to investigate the biochemical events that occur over time after thawing to optimize transfusion timing and evaluate the effect of platelet supernatants on tumor cell behavior in vitro. We compared fresh (Fresh-PLT) with Cryopreserved platelets (Cryo-PLT) at 1 h, 3 h and 6 h after thawing. MCF-7 and HL-60 cells were cultured with Fresh- or 1 h Cryo-PLT supernatants to investigate cell proliferation, migration, and PLT-cell adhesion. We noticed a significant impairment of hemostatic activity accompanied by a post-thaw decrease of CD42b+ , which identifies the CD62P--population. FTIR spectroscopy revealed a decrease in the total protein content together with changes in their conformational structure, which identified two sub-groups: 1) Fresh and 1 h Cryo-PLT; 2) 3 h and 6 h cryo-PLT. Extracellular vesicle shedding and phosphatidylserine externalization (PS) increased after thawing. Cryo-PLT supernatants inhibited cell proliferation, impaired MCF-7 cell migration, and reduced ability to adhere to tumor cells. Within the first 3 hours after thawing, irreversible alterations of biomolecular structure occur in Cryo-PLT. Nevertheless, Cryo-PLT should be considered safe for the transfusion of cancer patients because of their insufficient capability to promote cancer cell proliferation, adhesion, or migration.

What is the context? Transfusion of Fresh platelets (Fresh-PLT) with prophylaxis purposes is common in onco-hematological patients.Cryopreservation is an alternative storage method that allows to extend platelet component shelf life and build supplies usable in case of emergency.It is well established that cryopreservation affects platelet function questioning their use in onco-hematological patients.It is still unknown how platelet impairment, induced by cryopreservation, occurs over time after thawing, nor how the by-products of PLT deterioration may impact on cancer cell behavior.What is new? In this study, we deeply characterized the functional and morphological changes induced by cryopreservation on platelets by comparing Fresh-PLT with Cryo-PLT at 1 h, 3 h and 6 h after thawing. Afterwards, we evaluated the effect of PLT supernatants on cancer cell behavior in vitro.The data presented show that within 3 hours after thawing Cryo-PLT undergo to irreversible macromolecular changes accompanied by increase of peroxidation processes and protein misfolding.After thawing the clot formation is reduced but still supported at all-time points measured, combined with unchanged phosphatidylserine expression and extracellular vesicles release over time.Cryo-PLT supernatants do not sustain proliferation and migration of cancer cells.WHAT is the impact? Cryo-PLT may be considered a precious back-up product to be used during periods of Fresh-PLT shortage to prevent bleeding in non-hemorrhagic patients.It is desirable to make it logistically feasible to transfuse cryopreserved platelets within 1 hour of thawing to maintain the platelets in their best performing condition.

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.

Human Neocortex Layer Features Evaluated by PIXE, STIM, and STXM Techniques.

The human neocortex has a cytoarchitecture composed of six layers with an intrinsic organization that relates to afferent and efferent pathways for a high functional specialization. Various histological, neurochemical, and connectional techniques have been used to study these cortical layers. Here, we explore the additional possibilities of swift ion beam and synchrotron radiation techniques to distinguish cellular layers based on the elemental distributions and areal density pattern in the human neocortex. Temporal cortex samples were obtained from two neurologically normal adult men (postmortem interval: 6-12 h). A cortical area of 500 × 500 µm2 was scanned by a 3 MeV proton beam for elemental composition and areal density measurements using particle induced x-ray emission (PIXE) and scanning transmission ion microscopy (STIM), respectively. Zinc showed higher values in cortical layers II and V, which needs a critical discussion. Furthermore, the areal density decreased in regions with a higher density of pyramidal neurons in layers III and V. Scanning transmission X-ray microscopy (STXM) revealed the cellular density with higher lateral resolution than STIM, but not enough to distinguish each cortical lamination border. Our data describe the practical results of these approaches employing both X-ray and ion-beam based techniques for the human cerebral cortex and its heterogeneous layers. These results add to the potential approaches and knowledge of the human neocortical gray matter in normal tissue to develop improvements and address further studies on pathological conditions.

X-rays induced alterations in mechanical and biochemical properties of isolated SH-SY5Y nuclei.

BACKGROUND: The use of ionizing radiations in radiotherapy is an effective and very common cancer treatment after surgery. Although ionizing-radiation DNA damages are extensively investigated, little is known about their effects on the other nuclear components, since their variations when studied in whole cells can be difficult to decouple from those of the cytoplasmatic structures. The organization of nuclear components plays a functional role since they are directly involved in some of the nuclear response to chemical or physical stimuli. For this reason, studying the X-ray effects on nuclear components is a crucial step in radiobiology. MATERIALS AND METHODS: We have used Atomic Force Microscopy (AFM) and micro-FTIR to examine the biomechanical and biochemical properties of hydrated fixed nuclei isolated from neuroblastoma (SH-SY5Y) cells irradiated by 2, 4, 6 and 8 Gy X-ray doses. RESULTS: The experimental results have shown that, already at 2 Gy irradiation dose, the nuclei exhibit not only a DNA damage, but also relevant alterations of lipid saturation, protein secondary structure arrangement and a significant decrease in nuclear stiffness, which indicate a remarkable chromatin decondensation. CONCLUSIONS AND GENERAL SIGNIFICANCE: The present work demonstrates that a multi-technique approach, able to disclose multiple features, can be helpful to achieve a comprehensive picture of the X-ray irradiation effects of the nuclear components and distinguish them from those occurring at the level of cytoplasm.

Subcellular elements responsive to the biomechanical activity of triple-negative breast cancer-derived small extracellular vesicles.

Triple-negative breast cancer (TNBC) stands out for its aggressive, fast spread, and highly metastatic behavior and for being unresponsive to the classical hormonal therapy. It is considered a disease with a poor prognosis and limited treatment options. Among the mechanisms that contribute to TNBC spreading, attention has been recently paid to small extracellular vesicles (sEVs), nano-sized vesicles that by transferring bioactive molecules to recipient cells play a crucial role in the intercellular communication among cancer, healthy cells, and tumor microenvironment. In particular, TNBC-derived sEVs have been shown to alter proliferation, metastasis, drug resistance, and biomechanical properties of target cells. To shed light on the molecular mechanisms involved in sEVs mediation of cell biomechanics, we investigated the effects of sEVs on the main subcellular players, i.e., cell membrane, cytoskeleton, and nuclear chromatin organization. Our results unveiled that TNBC-derived sEVs are able to promote the formation and elongation of cellular protrusions, soften the cell body, and induce chromatin decondensation in recipient cells. In particular, our data suggest that chromatin decondensation is the main cause of the global cell softening. The present study added new details and unveiled a novel mechanism of activity of the TNBC-derived sEVs, providing information for the efficient translation of sEVs to cancer theranostics.

Polyvinylidene Fluoride Aerogels with Tailorable Crystalline Phase Composition.

In this work, polyvinylidene fluoride (PVDF) aerogels with a tailorable phase composition were prepared by following the crystallization-induced gelation principle. A series of PVDF wet gels (5 to 12 wt.%) were prepared from either PVDF−DMF solutions or a mixture of DMF and ethanol as non-solvent. The effects of the non-solvent concentration on the crystalline composition of the PVDF aerogels were thoroughly investigated. It was found that the nucleating role of ethanol can be adjusted to produce low-density PVDF aerogels, whereas the changes in composition by the addition of small amounts of water to the solution promote the stabilization of the valuable ß and γ phases. These phases of the aerogels were monitored by FTIR and Raman spectroscopies. Furthermore, the crystallization process was followed by in-time and in situ ATR−FTIR spectroscopy. The obtained aerogels displayed specific surface areas > 150 m2 g−1, with variable particle morphologies that are dependent on the non-solvent composition, as observed by using SEM and Synchrotron Radiation Computed micro-Tomography (SR-µCT).

Study of the Spatio-Chemical Heterogeneity of Tannin-Furanic Foams: From 1D FTIR Spectroscopy to 3D FTIR Micro-Computed Tomography.

Tannin-furanic rigid foams are bio-based copolymers of tannin plant extract and furfuryl alcohol, promising candidates to replace synthetic insulation foams, as for example polyurethanes and phenolics, in eco-sustainable buildings thanks to their functional properties, such as lightness of the material and fire resistance. Despite their relevance as environmental-friendly alternatives to petroleum derivatives, many aspects of the polymerization chemistry still remain unclear. One of the open issues is on the spatial heterogeneity of the foam, i.e., whether the foam constituents prevalently polymerize in spatially segregated blocks or distribute almost homogenously in the foam volume. To address this matter, here we propose a multiscale FTIR study encompassing 1D FTIR spectroscopy, 2D FTIR imaging and 3D FTIR micro-tomography (FTIR-µCT) on tannin-furanic rigid foams obtained by varying the synthesis parameters in a controlled way. Thanks to the implementation of the acquisition and processing pipeline of FTIR-µCT, we were able for the first time to demonstrate that the polymer formulations influence the spatial organization of the foam at the microscale and, at the same time, prove the reliability of FTIR-µCT data by comparing 2D FTIR images and the projection of the 3D chemical images on the same plane.

ATR-Spin: an open-source 3D printed device for direct cytocentrifugation onto attenuated total reflectance crystals.

Infrared spectroscopy (IR) enables the direct and rapid characterization of cells at the molecular level. Achieving a rapid and consistent cell preparation is critical for the development of point-of-care diagnostics for cell analysis. Here we introduce an open-source, 3D printed device for integrating the isolation, preconcentration, and measurement of attenuated total reflectance IR spectra of cells from biofluids. The tool comprises a disposable card for cytocentrifugation, equipped with magnets, which allows reproducible integration into the pathlength of the IR spectrophotometer. Preliminary results using cell culture media containing A549 cells indicate that this system enables a qualitative and quantitative characterization of cells down to 10 cells µL-1 by using a single and cost-effective device and within a few minutes.

Efficient long-range conduction in cable bacteria through nickel protein wires.

Filamentous cable bacteria display long-range electron transport, generating electrical currents over centimeter distances through a highly ordered network of fibers embedded in their cell envelope. The conductivity of these periplasmic wires is exceptionally high for a biological material, but their chemical structure and underlying electron transport mechanism remain unresolved. Here, we combine high-resolution microscopy, spectroscopy, and chemical imaging on individual cable bacterium filaments to demonstrate that the periplasmic wires consist of a conductive protein core surrounded by an insulating protein shell layer. The core proteins contain a sulfur-ligated nickel cofactor, and conductivity decreases when nickel is oxidized or selectively removed. The involvement of nickel as the active metal in biological conduction is remarkable, and suggests a hitherto unknown form of electron transport that enables efficient conduction in centimeter-long protein structures.

Infrared Based Saliva Screening Test for COVID-19.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has resulted in an unprecedented need for diagnostic testing that is critical in controlling the spread of COVID-19. We propose a portable infrared spectrometer with purpose-built transflection accessory for rapid point-of-care detection of COVID-19 markers in saliva. Initially, purified virion particles were characterized with Raman spectroscopy, synchrotron infrared (IR) and AFM-IR. A data set comprising 171 transflection infrared spectra from 29 subjects testing positive for SARS-CoV-2 by RT-qPCR and 28 testing negative, was modeled using Monte Carlo Double Cross Validation with 50 randomized test and model sets. The testing sensitivity was 93 % (27/29) with a specificity of 82 % (23/28) that included positive samples on the limit of detection for RT-qPCR. Herein, we demonstrate a proof-of-concept high throughput infrared COVID-19 test that is rapid, inexpensive, portable and utilizes sample self-collection thus minimizing the risk to healthcare workers and ideally suited to mass screening.

Oxidation of ultralene and paraffin due to radiation damage after exposure to soft X-rays probed by FTIR microspectroscopy and X-ray fluorescence.

Radiation damage upon soft X-ray exposure is an important issue to be considered in soft X-ray microscopy. The work presented here is part of a more extended study on the topic and focuses on the effects of soft X-rays on paraffin, a common embedding medium for soft-tissues, and on ultralene and Si3N4 windows as sample supports. Our studies suggest that the sample environment indeed plays an important role in the radiation damage process and therefore should be carefully taken into account for the analysis and interpretation of new data. The radiation damage effects were followed over time using a combination of Fourier transform infrared (FTIR) microspectroscopy and X-ray fluorescence (XRF), and it was demonstrated that, for higher doses, an oxidation of both embedding medium and ultralene substrate takes place after the irradiated sample is exposed to air. This oxidation is reflected in a clear increase of C=O and O-H infrared bands and on the XRF oxygen maps, correlated with a decrease of the aliphatic infrared signal. The results also show that the oxidation process may affect quantitative evaluation of light element concentrations.

Infrared Spectroscopy of Blood.

The magnitude of infectious diseases in the twenty-first century created an urgent need for point-of-care diagnostics. Critical shortages in reagents and testing kits have had a large impact on the ability to test patients with a suspected parasitic, bacteria, fungal, and viral infections. New point-of-care tests need to be highly sensitive, specific, and easy to use and provide results in rapid time. Infrared spectroscopy, coupled to multivariate and machine learning algorithms, has the potential to meet this unmet demand requiring minimal sample preparation to detect both pathogenic infectious agents and chronic disease markers in blood. This focal point article will highlight the application of Fourier transform infrared spectroscopy to detect disease markers in blood focusing principally on parasites, bacteria, viruses, cancer markers, and important analytes indicative of disease. Methodologies and state-of-the-art approaches will be reported and potential confounding variables in blood analysis identified. The article provides an up to date review of the literature on blood diagnosis using infrared spectroscopy highlighting the recent advances in this burgeoning field.

Assessing the impacts of sewage sludge amendment containing nano-TiO2 on tomato plants: A life cycle study.

Increasing evidence indicates the presence of engineered nanoparticles (ENPs) in sewage sludge derived from wastewater treatment. Land application of sewage sludge is, therefore, considered as an important pathway for ENP transfer to the environment. The aim of this work was to understand the effects of sewage sludge containing nano-TiO2 on plants (tomato) when used as an amendment in agricultural soil. We assessed developmental parameters for the entire plant life cycle along with metabolic and bio-macromolecule changes and titanium accumulation in plants. The results suggest that the sewage sludge amendment containing nano-TiO2 increased plant growth (142% leaf biomass, 102% fruit yield), without causing changes in biochemical responses, except for a 43% decrease in leaf tannin concentration. Changes in elemental concentrations (mainly Fe, B, P, Na, and Mn) of plant stem, leaves and, to a lesser extent fruits were observed. Fourier-transformed infrared analysis showed maximum changes in plant leaves (decrease in tannins and lignins and increase in carbohydrates) but no change in fruits. No significant Ti enrichment was detected in tomato fruits. In conclusion, we evidenced no acute toxicity to plants and no major implication for food safety after one plant life cycle exposure.

Micro-ATR FTIR, SEM-EDS and X-ray micro-CT: an innovative multi-technique approach to investigate bone affected by peri-implantitis.

PURPOSE: The aim of this work was to prove the synergic complementarity of attenuated total reflection Fourier transform infrared microspectroscopy (micro-ATR FTIR), scanning electron microscopy (SEM) coupled with energy-dispersive x-ray spectroscopy (EDS) and x-ray computed microtomography (micro-CT) by studying implant samples with bone affected by peri-implantitis. MATERIALS AND METHODS: Six samples of implanted bone affected by peri-implantitis and one control healthy bone were analyzed. Thick bone sections included in epoxy-resin and removed implants were analyzed by micro-ATR FTIR, SEM-EDS, and micro-CT. RESULTS: Micro-ATR FTIR revealed the complex nature of the bone composition. Vibrational bands characteristic of both mineral bone phase (acidic phosphates, CO32- groups) and organic bone phase (mostly collagen) could be recognized, and their proportion could be seen to change accordingly with the bone degradation. Similarly, SEM-EDS clearly revealed the cortical nature of the control mandible and its homogenous mineral composition. On the contrary, EDS analyses performed over relevant portions of pathologic samples revealed that defective areas were almost Ca and P free. Micro-CT data showed that the morphology of the interface was smooth and linear in the physiologic periimplant bone, while in the pathologic samples, an altered morphology was evident. CONCLUSION: This study demonstrated that morphologic, elemental, and biochemical modifications of periimplant bone can be studied using micro-ATR FTIR, SEM-EDS, and micro-CT. The complement of these techniques can be considered a new multipurpose approach to investigate bone affected by peri-implantitis.

Zinc incorporation in marine bivalve shells grown in mine-polluted seabed sediments: a case study in the Malfidano mining area (SW Sardinia, Italy).

Zinc incorporation into marine bivalve shells belonging to different genera (Donax, Glycymeris, Lentidium, and Chamelea) grown in mine-polluted seabed sediments (Zn up to 1% w/w) was investigated using x-ray diffraction (XRD), chemical analysis, soft x-ray microscopy combined with low-energy x-ray fluorescence (XRF) mapping, x-ray absorption spectroscopy (XAS), and transmission electron microscopy (TEM). These bivalves grew their shells, producing aragonite as the main biomineral and they were able to incorporate up to 2.0-80 mg/kg of Zn, 5.4-60 mg/kg of Fe and 0.5-4.5 mg/kg of Mn. X-ray absorption near edge structure (XANES) analysis revealed that for all the investigated genera, Zn occurred as independent Zn mineral phases, i.e., it was not incorporated or adsorbed into the aragonitic lattice. Overall, our results indicated that Zn coordination environment depends on the amount of incorporated Zn. Zn phosphate was the most abundant species in Donax and Lentidium genera, whereas, Chamelea shells, characterized by the highest Zn concentrations, showed the prevalence of Zn-cysteine species (up to 56% of total speciation). Other Zn coordination species found in the investigated samples were Zn hydrate carbonate (hydrozincite) and Zn phosphate. On the basis of the coordination environments, it was deduced that bivalves have developed different biogeochemical mechanisms to regulate Zn content and its chemical speciation and that cysteine plays an important role as an active part of detoxification mechanism. This work represents a step forward for understanding bivalve biomineralization and its significance for environmental monitoring and paleoreconstruction.

In vitro FTIR microspectroscopy analysis of primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil: a new spectroscopic approach for studying the drug-cell interaction.

In the present study, human primary oral squamous carcinoma cells treated with cisplatin and 5-fluorouracil were analyzed, for the first time, by in vitro FTIR Microspectroscopy (FTIRM), to improve the knowledge on the biochemical pathways activated by these two chemotherapy drugs. To date, most of the studies regarding FTIRM cellular analysis have been executed on fixed cells from immortalized cell lines. FTIRM analysis performed on primary tumor cells under controlled hydrated conditions provides more reliable information on the biochemical processes occurring in in vivo tumor cells. This spectroscopic analysis allows to get on the same sample and at the same time an overview of the composition and structure of the most remarkable cellular components. In vitro FTIRM analysis of primary oral squamous carcinoma cells evidenced a time-dependent drug-specific cellular response, also including apoptosis triggering. Furthermore, the univariate and multivariate analyses of IR data evidenced meaningful spectroscopic differences ascribable to alterations affecting cellular proteins, lipids and nucleic acids. These findings suggest for the two drugs different pathways and extents of cellular damage, not provided by conventional cell-based assays (MTT assay and image-based cytometry).

Light element distribution in fresh and frozen-thawed human ovarian tissues: a preliminary study.

RESEARCH QUESTION: Does synchrotron X-ray fluorescence (XRF) provide novel chemical information for the evaluation of human ovarian tissue cryopreservation protocols? DESIGN: Tissues from five patients undergoing laparoscopic surgery for benign gynaecological conditions were fixed for microscopic analysis either immediately or after cryopreservation. After fixation, fresh and slowly frozen samples were selected by light microscopy and transmission electron microscopy, and subsequently analysed with synchrotron XRF microscopy at different incident energies. RESULTS: The distributions of elements detected at 7.3 keV (S, P, K, Cl, Fe, and Os) and 1.5 keV (Na and Mg) were related to the changes revealed by light microscopy and transmission electron microscopy analyses. The light elements showed highly informative findings. The S distribution was found to be an indicator of extracellular component changes in the stromal tissues of the freeze-stored samples, further revealed by the transmission electron microscopy analyses. Low-quality follicles, frequent in the freeze-thawed tissues, showed a high Na level in the ooplasm. On the contrary, good-quality follicles were detected by a homogeneous Cl distribution. The occurrence of vacuolated follicles increased after cryopreservation, and the XRF analyses showed that the vacuolar structures contained mainly Cl and Na. CONCLUSIONS: The study demonstrates that elemental imaging techniques, particularly revealing the distribution of light elements, could be useful in establishing new cryopreservation protocols.

Effects of soft X-ray radiation damage on paraffin-embedded rat tissues supported on ultralene: a chemical perspective.

Radiation damage is an important aspect to be considered when analysing biological samples with X-ray techniques as it can induce chemical and structural changes in the specimens. This work aims to provide new insights into the soft X-ray induced radiation damage of the complete sample, including not only the biological tissue itself but also the substrate and embedding medium, and the tissue fixation procedure. Sample preparation and handling involves an unavoidable interaction with the sample matrix and could play an important role in the radiation-damage mechanism. To understand the influence of sample preparation and handling on radiation damage, the effects of soft X-ray exposure at different doses on ultralene, paraffin and on paraffin-embedded rat tissues were studied using Fourier-transform infrared (FTIR) microspectroscopy and X-ray microscopy. Tissues were preserved with three different commonly used fixatives: formalin, glutaraldehyde and Karnovsky. FTIR results showed that ultralene and paraffin undergo a dose-dependent degradation of their vibrational profiles, consistent with radiation-induced oxidative damage. In addition, formalin fixative has been shown to improve the preservation of the secondary structure of proteins in tissues compared with both glutaraldehyde and Karnovsky fixation. However, conclusive considerations cannot be drawn on the optimal fixation protocol because of the interference introduced by both substrate and embedding medium in the spectral regions specific to tissue lipids, nucleic acids and carbohydrates. Notably, despite the detected alterations affecting the chemical architecture of the sample as a whole, composed of tissue, substrate and embedding medium, the structural morphology of the tissues at the micrometre scale is essentially preserved even at the highest exposure dose.

Pitfalls and promises in FTIR spectromicroscopy analyses to monitor iron-mediated DNA damage in sperm.

Many drugs, chemicals, and environmental factors can impair sperm functionality by inducing DNA damage, one of the important causes of reduced fertility potential. The use of vibrational spectromicroscopy represents a promising approach for monitoring DNA integrity in sperm, although some limitations exist, depending from the experimental conditions. Here, we report that when using FTIR spectromicroscopy to reveal oxidative stress mediated by Fenton's reaction on hydrated sperm samples, DNA damage interpretation is partially compromised by unexpected cell surface precipitates. The precipitates give a broad band in the 1150-1000cm(-1) infrared region, which partially covers one of the signatures of DNA (phosphate stretching bands), and are detected as iron and oxygen containing material when using XRF spectroscopy. On the other hand, the analyses further support the potential of FTIR spectromicroscopy to reveal cellular oxidative damage events such as lipid peroxidation, protein misfolding and aggregations, as well as DNA strain breaks.