Small-Angle X-ray Scattering (SAXS) Used for the Identification of Nicomorphine Polymorphic Changes at the Early Stage to Avoid Varied Stability and Possible Side Effects.

In this paper, we present the identification of polymorphisms at an early stage, identified by applying non-standard methods such as SAXS. We provide an analytical approach to polymorphism in the quality/purity of an active pharmaceutical ingredient (API), supplied to a generic company by two different suppliers (i.e., manufacturers). Changes in thermodynamic polymorphism firstly become visible in traces in the larger crystal lattices, which are visible on the SAXS spectrum only using the logarithmic scale, as shown in the result figures. Hence, we are here on the trail of the beginning of a new polymorph in nicomorphine, whose crystal waviness at the early stage is visible only in the additional symmetrical peaks identified and calculated using SAXS, while the chemical analyses excluded all kinds of chemical impurities. The chemical and structural properties were studied using the following techniques: SAXS, WAXS, DSC, dissolution, Raman spectroscopy, and FTIR. Only the SAXS technique could identify crucial differences and calculate the additional signals related to giant crystals, whilst a standard method such as WAXS showed none, and nor did the chemical analyses, such as Raman spectroscopy and FT-IR. This means that due to water in crystallization (known in nicomorphine) or thermodynamic waviness, the formation of the new polymorph starts first in traces, which become visible at larger distances from the crystal lattice, detectible only in the SAXS range. This is a very important premise and hypothesis for further research, and we believe that this work lays a new stone in understanding the origin of new unknown polymorphs and their mixtures. Therefore, the aim of this work is to show that the use of non-standard methods (i.e., SAXS) can be of great benefit to API analysis and the identification of polymorphic changes in the early phase, which can cause varied stability, solubility and bioavailability and thus different therapeutic effects or side effects.

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.

Morphological and lipid metabolism alterations in macrophages exposed to model environmental nanoplastics traced by high-resolution synchrotron techniques.

The release of nanoplastics (NPs) in the environment is a significant health concern for long-term exposed humans. Although their usage has certainly revolutionized several application fields, at nanometer size, NPs can easily interact at the cellular level, resulting in potential harmful effects. Micro/Nanoplastics (M/NPs) have a demonstrated impact on mammalian endocrine components, such as the thyroid, adrenal gland, testes, and ovaries, while more investigations on prenatal and postnatal exposure are urgently required. The number of literature studies on the NPs' presence in biological samples is increasing. However, only a few offer a close study on the model environmental NP-immune system interaction exploited by advanced microscopy techniques. The present study highlights substantial morphological and lipid metabolism alterations in human M1 macrophages exposed to labeled polypropylene and polyvinyl chloride nanoparticles (PP and PVC NPs) (20 µg/ml). The results are interpreted by advanced microscopy techniques combined with standard laboratory tests and fluorescence microscopy. We report the accurate detection of polymeric nanoparticles doped with cadmium selenide quantum dots (CdSe-QDs NPs) by following the Se (L line) X-ray fluorescence emission peak at higher sub-cellular resolution, compared to the supportive light fluorescence microscopy. In addition, scanning transmission X-ray microscopy (STXM) imaging successfully revealed morphological changes in NP-exposed macrophages, providing input for Fourier transform infrared (FTIR) spectroscopy analyses, which underlined the chemical modifications in macromolecular components, specifically in lipid response. The present evidence was confirmed by quantifying the lipid droplet (LD) contents in PP and PVC NPs-exposed macrophages (0-100 µg/ml) by Oil Red O staining. Hence, even at experimental NPs' concentrations and incubation time, they do not significantly affect cell viability; they cause an evident lipid metabolism impairment, a hallmark of phagocytosis and oxidative stress.

Cadmium Sulfide Quantum Dots, Mitochondrial Function and Environmental Stress: A Mechanistic Reconstruction through In Vivo Cellular Approaches in Saccharomyces cerevisiae.

Research on the effects of engineered nanomaterials (ENMs) on mitochondria, which represent one of the main actors in cell function, highlighted effects on ROS production, gametogenesis and organellar genome replication. Specifically, the mitochondrial effects of cadmium sulfide quantum dots (CdS QDs) exposure can be observed through the variation in enzymatic kinetics at the level of the respiratory chain and also by analyzing modifications of reagent and products in term of the bonds created and disrupted during the reactions through Fourier-transform infrared spectroscopy (FTIR). This study investigated both in intact cells and in isolated mitochondria to observe the response to CdS QDs treatment at the level of electron transport chain in the wild-type yeast Saccharomyces cerevisiae and in the deletion mutant Δtom5, whose function is implicated in nucleo-mitochondrial protein trafficking. The changes observed in wild type and Δtom5 strains in terms of an increase or decrease in enzymatic activity (ranging between 1 and 2 folds) also differed according to the genetic background of the strains and the respiratory chain functionality during the CdS QDs treatment performed. Results were confirmed by FTIR, where a clear difference between the QD effects in the wild type and in the mutant strain, Δtom5, was observed. The utilization of these genetic and biochemical approaches is instrumental to clarify the mitochondrial mechanisms implicated in response to these types of ENMs and to the stress response that follows the exposure.

Infrared nanospectroscopy depth-dependent study of modern materials: morpho-chemical analysis of polyurethane/fibroin binary meshes.

Infrared scattering-type scanning near-field optical microscopy (IR s-SNOM) and imaging is here exploited together with attenuated total reflection (ATR) IR imaging and scanning electron microscopy (SEM) to depict the chemical composition of fibers in hybrid electrospun meshes. The focus is on a recently developed bio-hybrid material for vascular tissue engineering applications, named Silkothane®, obtained in the form of nanofibrous matrices from the processing of a silk fibroin-polyurethane (SFPU) blend via electrospinning. Morphology and chemistry of single fibers, at both surface and subsurface level, have been successfully characterized with nanoscale resolution, taking advantage of the IR s-SNOM capability to portray the nanoscale depth profile of this modern material working at diverse harmonics of the signal. The applied methodology allowed to describe the superficial characteristics of the mesh up to a depth of about 100 nm, showing that SF and PU do not tend to co-aggregate to form hybrid fibers, at least at the length scale of hundreds of nanometers, and that subdomains other than the fibrillar ones can be present. More generally, in the present contribution, the depth profiling capabilities of IR s-SNOM, so far theoretically predicted and experimentally proven only on model systems, have been corroborated on a real material in its natural conditions with respect to production, opening the room for the exploitation of IR s-SNOM as valuable technique to support the production and the engineering of nanostructured materials by the precise understanding of their chemistry at the interface with the environment.

Variabilities in global DNA methylation and ß-sheet richness establish spectroscopic landscapes among subtypes of pancreatic cancer.

PURPOSE: Knowledge about pancreatic cancer (PC) biology has been growing rapidly in recent decades. Nevertheless, the survival of PC patients has not greatly improved. The development of a novel methodology suitable for deep investigation of the nature of PC tumors is of great importance. Molecular imaging techniques, such as Fourier transform infrared (FTIR) spectroscopy and Raman hyperspectral mapping (RHM) combined with advanced multivariate data analysis, were useful in studying the biochemical composition of PC tissue. METHODS: Here, we evaluated the potential of molecular imaging in differentiating three groups of PC tumors, which originate from different precursor lesions. Specifically, we comprehensively investigated adenocarcinomas (ACs): conventional ductal AC, intraductal papillary mucinous carcinoma, and ampulla of Vater AC. FTIR microspectroscopy and RHM maps of 24 PC tissue slides were obtained, and comprehensive advanced statistical analyses, such as hierarchical clustering and nonnegative matrix factorization, were performed on a total of 211,355 Raman spectra. Additionally, we employed deep learning technology for the same task of PC subtyping to enable automation. The so-called convolutional neural network (CNN) was trained to recognize spectra specific to each PC group and then employed to generate CNN-prediction-based tissue maps. To identify the DNA methylation spectral markers, we used differently methylated, isolated DNA and compared the observed spectral differences with the results obtained from cellular nuclei regions of PC tissues. RESULTS: The results showed significant differences among cancer tissues of the studied PC groups. The main findings are the varying content of ß-sheet-rich proteins within the PC cells and alterations in the relative DNA methylation level. Our CNN model efficiently differentiated PC groups with 94% accuracy. The usage of CNN in the classification task did not require Raman spectral data preprocessing and eliminated the need for extensive knowledge of statistical methodologies. CONCLUSIONS: Molecular spectroscopy combined with CNN technology is a powerful tool for PC detection and subtyping. The molecular fingerprint of DNA methylation and ß-sheet cytoplasmic proteins established by our results is different for the main PC groups and allowed the subtyping of pancreatic tumors, which can improve patient management and increase their survival. Our observations are of key importance in understanding the variability of PC and allow translation of the methodology into clinical practice by utilizing liquid biopsy testing.

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.

Unraveling the timescale of the structural photo-response within oriented metal-organic framework films.

Fundamental knowledge on the intrinsic timescale of structural transformations in photo-switchable metal-organic framework films is crucial to tune their switching performance and to facilitate their applicability as stimuli-responsive materials. In this work, for the first time, an integrated approach to study and quantify the temporal evolution of structural transformations is demonstrated on an epitaxially oriented DMOF-1-on-MOF film system comprising azobenzene in the DMOF-1 pores (DMOF-1/AB). We employed time-resolved Grazing Incidence Wide-Angle X-Ray Scattering measurements to track the structural response of the DMOF-1/AB film upon altering the length of the azobenzene molecule by photo-isomerization (trans-to-cis, 343 nm; cis-to-trans, 450 nm). Within seconds, the DMOF-1/AB response occurred fully reversible and over several switching cycles by cooperative photo-switching of the oriented DMOF-1/AB crystallites as confirmed further by infrared measurements. Our work thereby suggests a new avenue to elucidate the timescales and photo-switching characteristics in structurally responsive MOF film systems.

A Nanofocused Light on Stradivari Violins: Infrared s-SNOM Reveals New Clues Behind Craftsmanship Mastery.

It is well-known that all the phases of the manufacturing influence the extraordinary aesthetic and acoustic features of Stradivari's instruments. However, these masterpieces still keep some of their secrets hidden by the lack of documentary evidence. In particular, there is not a general consensus on the use of a protein-based ground coating directly spread on the wood surface by the Cremonese Master. The present work demonstrates that infrared scattering-type scanning near-fields optical microscopy (s-SNOM) may provide unprecedented information on very complex cross-sectioned microsamples collected from two of Stradivari's violins, nanoresolved chemical sensitivity being the turning point for detecting minute traces of a specific compound, namely proteins, hidden by the matrix when macro or micro sampling approaches are exploited. This nanoresolved chemical-sensitive technique contributed new and robust evidence to the long-debated question about the use of proteinaceous materials by Stradivari.

Oleic Acid Protects Endothelial Cells from Silica-Coated Superparamagnetic Iron Oxide Nanoparticles (SPIONs)-Induced Oxidative Stress and Cell Death.

Superparamagnetic iron oxide nanoparticles (SPIONs) have great potential for use in medicine, but they may cause side effects due to oxidative stress. In our study, we investigated the effects of silica-coated SPIONs on endothelial cells and whether oleic acid (OA) can protect the cells from their harmful effects. We used viability assays, flow cytometry, infrared spectroscopy, fluorescence microscopy, and transmission electron microscopy. Our results show that silica-coated SPIONs are internalized by endothelial cells, where they increase the amount of reactive oxygen species (ROS) and cause cell death. Exposure to silica-coated SPIONs induced accumulation of lipid droplets (LD) that was not dependent on diacylglycerol acyltransferase (DGAT)-mediated LD biogenesis, suggesting that silica-coated SPIONs suppress LD degradation. Addition of exogenous OA promoted LD biogenesis and reduced SPION-dependent increases in oxidative stress and cell death. However, exogenous OA protected cells from SPION-induced cell damage even in the presence of DGAT inhibitors, implying that LDs are not required for the protective effect of exogenous OA. The molecular phenotype of the cells determined by Fourier transform infrared spectroscopy confirmed the destructive effect of silica-coated SPIONs and the ameliorative role of OA in the case of oxidative stress. Thus, exogenous OA protects endothelial cells from SPION-induced oxidative stress and cell death independent of its incorporation into triglycerides.

Effects of Ionizing Radiation and Long-Term Storage on Hydrated vs. Dried Cell Samples of Extremophilic Microorganisms.

A main factor hampering life in space is represented by high atomic number nuclei and energy (HZE) ions that constitute about 1% of the galactic cosmic rays. In the frame of the "STARLIFE" project, we accessed the Heavy Ion Medical Accelerator (HIMAC) facility of the National Institute of Radiological Sciences (NIRS) in Chiba, Japan. By means of this facility, the extremophilic species Haloterrigena hispanica and Parageobacillus thermantarcticus were irradiated with high LET ions (i.e., Fe, Ar, and He ions) at doses corresponding to long permanence in the space environment. The survivability of HZE-treated cells depended upon either the storage time and the hydration state during irradiation; indeed, dry samples were shown to be more resistant than hydrated ones. With particular regard to spores of the species P. thermantarcticus, they were the most resistant to irradiation in a water medium: an analysis of the changes in their biochemical fingerprinting during irradiation showed that, below the survivability threshold, the spores undergo to a germination-like process, while for higher doses, inactivation takes place as a consequence of the concomitant release of the core's content and a loss of integrity of the main cellular components. Overall, the results reported here suggest that the selected extremophilic microorganisms could serve as biological model for space simulation and/or real space condition exposure, since they showed good resistance to ionizing radiation exposure and were able to resume cellular growth after long-term storage.

Live-Cell Synchrotron-Based FTIR Evaluation of Metabolic Compounds in Brain Glioblastoma Cell Lines after Riluzole Treatment.

Glioblastoma multiforme (GBM) is the most aggressive brain tumor, characterized by short median survival and an almost 100% tumor-related mortality. The standard of care treatment for newly diagnosed GBM includes surgical resection followed by concomitant radiochemotherapy. The prevention of disease progression fails due to the poor therapeutic effect caused by the great molecular heterogeneity of this tumor. Previously, we exploited synchrotron radiation-based soft X-ray tomography and hard X-ray fluorescence for elemental microimaging of the shock-frozen GBM cells. The present study focuses instead on the biochemical profiling of live GBM cells and provides new insight into tumor heterogenicity. We studied bio-macromolecular changes by exploring the live-cell synchrotron-based Fourier transform infrared (SR-FTIR) microspectroscopy in a set of three GBM cell lines, including the patient-derived glioblastoma cell line, before and after riluzole treatment, a medicament with potential anticancer properties. SR-FTIR microspectroscopy shows that GBM live cells of different origins recruit different organic compounds. The riluzole treatment of all GBM cell lines mainly affected carbohydrate metabolism and the DNA structure. Lipid structures and protein secondary conformation are affected as well by the riluzole treatment: cellular proteins assumed cross ß-sheet conformation while parallel ß-sheet conformation was less represented for all GBM cells. Moreover, we hope that a new live-cell approach for GBM simultaneous treatment and examination can be devised to target cancer cells more specifically, i.e., future therapies can develop more specific treatments according to the specific bio-macromolecular signature of each tumor type.

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.

Addressable graphene encapsulation of wet specimens on a chip for optical, electron, infrared and X-ray based spectromicroscopy studies.

Label-free spectromicroscopy methods offer the capability to examine complex cellular phenomena. Electron and X-ray based spectromicroscopy methods, though powerful, have been hard to implement with hydrated objects due to the vacuum incompatibility of the samples and due to the parasitic signals from (or drastic attenuation by) the liquid matrix surrounding the biological object of interest. Similarly, for many techniques that operate at ambient pressure, such as Fourier transform infrared spectromicroscopy (FTIRM), the aqueous environment imposes severe limitations due to the strong absorption of liquid water in the infrared regime. Here we propose a microfabricated multi-compartmental and reusable hydrated sample platform suitable for use with several analytical techniques, which employs the conformal encapsulation of biological specimens by a few layers of atomically thin graphene. Such an electron, X-ray, and infrared transparent, molecularly impermeable and mechanically robust enclosure preserves the hydrated environment around the object for a sufficient time to allow in situ examination of hydrated bio-objects with techniques operating under both ambient and high vacuum conditions. An additional hydration source, provided by hydrogel pads lithographically patterned in the liquid state near/around the specimen and co-encapsulated, has been added to further extend the hydration lifetime. Note that the in-liquid lithographic electron beam-induced gelation procedure allows for addressable capture and immobilization of the biological cells from the solution. Scanning electron microscopy and optical fluorescence microscopy, as well as synchrotron radiation based FTIR and X-ray fluorescence microscopy, have been used to test the applicability of the platform and for its validation with yeast, A549 human carcinoma lung cells and micropatterned gels as biological object phantoms.

Chemical constitution of polyfurfuryl alcohol investigated by FTIR and Resonant Raman spectroscopy.

The actual chemical structure of polyfurfuryl alcohol (PFA) is still uncertain in spite of several studies on the topic, variations during the polymerization processes being one reason that must be addressed. The use of a limited set of analytical techniques is often insufficient to provide an exhaustive chemical characterization. Moreover, it is still not possible to exactly determine presence and amount of each specific functional group in the polymeric structure. We employed both Fourier Transform Infrared Spectroscopy (FTIR) and Resonant Raman spectroscopy (RR), corroborated by quantum mechanically aided analysis of the experimental spectra, to infer about the chemical structure of two samples of PFAs, synthetized in different ways and appearing macroscopically different, the first one being a liquid and viscous commercial sample, the second one being a self-prepared solid and rigid sample produced following a thermosetting procedure. The vibrational spectroscopic analysis confirms the presence of differences in their chemical structures. The viscous form of PFA is mainly composed by short polymeric chains, and is characterized by the presence of isolated furfuryl alcohol and furfural residues similar to 5-hydroxymethylfurfural; the thermosetted PFA is formed by more cross-linked structures, characterized by several ketones and alkene double bonds, as well as a significant presence of Diels-Alder structures. In summary, the present study evidences how the use of both FTIR and RR spectroscopy, the latter carried out at several laser excitation wavelengths, indicates an accurate way to spectroscopically investigate complex polymers enabling to satisfactorily infer about their peculiar chemical structure.

Infrared Nanospectroscopy Reveals DNA Structural Modifications upon Immobilization onto Clay Nanotubes.

The growing demand for innovative means in biomedical, therapeutic and diagnostic sciences has led to the development of nanomedicine. In this context, naturally occurring tubular nanostructures composed of rolled sheets of alumino-silicates, known as halloysite nanotubes, have found wide application. Halloysite nanotubes indeed have surface properties that favor the selective loading of biomolecules. Here, we present the first, to our knowledge, structural study of DNA-decorated halloysite nanotubes, carried out with nanometric spatially-resolved infrared spectroscopy. Single nanotube absorption measurements indicate a partial covering of halloysite by DNA molecules, which show significant structural modifications taking place upon loading. The present study highlights the constraints for the use of nanostructured clays as DNA carriers and demonstrates the power of super-resolved infrared spectroscopy as an effective and versatile tool for the evaluation of immobilization processes in the context of drug delivery and gene transfer.

Binding of tyrosine kinase inhibitor to epidermal growth factor receptor: surface-enhanced infrared absorption microscopy reveals subtle protein secondary structure variations.

Surface-Enhanced Infrared Absorption (SEIRA) has been proposed as a valuable tool for protein binding studies, but its performances have been often proven on model proteins undergoing severe secondary structure rearrangements, while ligand binding only marginally involves the protein backbone in the vast majority of the biologically relevant cases. In this study we demonstrate the potential of SEIRA microscopy for highlighting the very subtle secondary structure modifications associated with the binding of Lapatinib, a tyrosine kinase inhibitor (TKI), to epidermal growth factor receptor (EGFR), a well-known driver of tumorigenesis in pathological settings such as lung, breast and brain cancers. By boosting the performances of Mid-IR plasmonic devices based on nanoantennas cross-geometry, accustoming the protein purification protocols, carefully tuning the protein anchoring methodology and optimizing the data analysis, we were able to detect EGFR secondary structure modification associated with few amino acids. A nano-patterned platform with this kind of sensitivity bridges biophysical and structural characterization methods, thus opening new possibilities in studying of proteins of biomedical interest, particularly for drug-screening purposes.

Plastics, (bio)polymers and their apparent biogeochemical cycle: An infrared spectroscopy study on foraminifera.

To understand the fate of plastic in oceans and the interaction with marine organisms, we investigated the incorporation of (bio)polymers and microplastics in selected benthic foraminiferal species by applying FTIR (Fourier Transform Infrared) microscopy. This experimental methodology has been applied to cultured benthic foraminifera Rosalina globularis, and to in situ foraminifera collected in a plastic remain found buried into superficial sediment in the Mediterranean seafloor, Rosalina bradyi, Textularia bocki and Cibicidoides lobatulus. In vitro foraminifera were treated with bis-(2-ethylhexyl) phthalate (DEHP) molecule to explore its internalization in the cytoplasm. Benthic foraminifera are marine microbial eukaryotes, sediment-dwelling, commonly short-lived and with reproductive cycles which play a central role in global biogeochemical cycles of inorganic and organic compounds. Despite the recent advances and investigations into the occurrence, distribution, and abundance of plastics, including microplastics, in marine environments, there remain relevant knowledge gaps, particularly on their effects on the benthic protists. No study, to our knowledge, has documented the molecular scale effect of plastics on foraminifera. Our analyses revealed three possible ways through which plastic-related molecules and plastic debris can enter a biogeochemical cycle and may affect the ecosystems: 1) foraminifera in situ can grow on plastic remains, namely C. lobatulus, R. bradyi and T. bocki, showing signals of oxidative stress and protein aggregation in comparison with R. globularis cultured in negative control; 2) DEHP can be incorporated in the cytoplasm of calcareous foraminifera, as observed in R. globularis; 3) microplastic debris, identified as epoxy resin, can be found in the cytoplasm and the agglutinated shell of T. bocki. We hypothesize that plastic waste and their associated additives may produce modifications related to the biomineralization process in foraminifera. This effect would be added to those induced by ocean acidification with negative consequences on the foraminiferal biogenic carbon (C) storage capacity.

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.

Cigarette butts, a threat for marine environments: Lessons from benthic foraminifera (Protista).

Cigarette butts are the most common form of litter in the world and their environmental impact is related to both persistence and potential toxic effects for chemical composition. The objective of this study was to assess the acute toxicity (LC50-48 h) of human-smoked cigarette butts leachate on 3 cultured genera of benthic foraminifera: the calcareous perforate Rosalina globularis, the calcareous imperforate Quinqueloculina spp., and the agglutinated Textularia agglutinans. The specimens were exposed to 16, 8, 4, 2, and 1 cigarette butts/L concentrations that prove to be acutely toxic to all taxa. Starting from 4 cigarette butts/L, both calcareous genera showed shell decalcification, and death of almost all the individuals, except for the more resistant agglutinated species. These results suggest the potential harmfulness of cigarette butts leachate related to pH reduction and release of toxic substances, in particular nicotine, which leads to physiology alteration and in many cases cellular death.