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Abstract Despite the extensive literature on the retrieval of digestible starches from archaeological contexts, there are still significant concerns regarding their genuine origin and durability. Here, we propose a multi-analytical strategy to identify the authenticity of ancient starches retrieved from macrolithic tools excavated at Upper Paleolithic sites in the Pontic steppe. This strategy integrates the morphological discrimination of starches through optical microscopy and scanning electron microscopy with single starch chemo-profiling using Fourier transform infrared imaging and microscopy. We obtained evidence of aging and biomineralization in the use-related starches from Palaeolithic sites, providing a methodology to establish their ancient origin, assess their preservation status, and attempt their identification. The pivotal application of this multidisciplinar approach demonstrates that the macrolithic tools, from which starches were dislodged, were used for food-processing across the Pontic Steppe around 40,000 years ago during the earliest colonization of Eurasia by Homo sapiens.

Abstract Coccolithophores, marine calcifying phytoplankton, are important primary producers impacting the global carbon cycle at different timescales. Their biomineral structures, the calcite containing coccoliths, are among the most elaborate hard parts of any organism. Understanding the morphogenesis of coccoliths is not only relevant in the context of coccolithophore eco-physiology but will also inform biomineralization and crystal design research more generally. The recent discovery of a silicon (Si) requirement for crystal shaping in some coccolithophores has opened up a new avenue of biomineralization research. In order to develop a mechanistic understanding of the role of Si, the presence and localization of this chemical element in coccoliths needs to be known. Here, we document for the first time the uneven Si distribution in Helicosphaera carteri coccoliths through three synchrotron-based techniques employing X-ray Fluorescence and Infrared Spectromicroscopy. The enrichment of Si in specific areas of the coccoliths point to a targeted role of this element in the coccolith formation. Our findings mark a key step in biomineralization research because it opens the door for a detailed mechanistic understanding of the role Si plays in shaping coccolith crystals.

Infrared spectro-microscopy is a powerful technique for analysing chemical maps of cells and tissues for biomedical and clinical applications, yet the strong water absorption in the mid-infrared region is a challenge to overcome, as it overlaps with the spectral fingerprints of biological components. Microfluidic chips offer ultimate control over the water layer thickness and are increasingly used in infrared spectro-microscopy. However, the actual impact of the water layer thickness on the instrument's performance is often left to the experimentalist's intuition and the peculiarities of specific instruments. Aiming to experimentally test the amount of absorption introduced by water with varying layer thicknesses, we fabricated a set of microfluidic devices with three controlled chamber thicknesses, each comprising a simple test pattern made of a well-known photoresist SU-8. We employed two infrared spectro-microscopy methods for measurements. The first method involves using a standard FTIR microscope with a benchtop infrared light source. The second method is a quantum infrared microscopy technique, where infrared imaging is achieved by detecting correlated photons in the visible range. We demonstrated that both methods enable the measurement of the absorption spectrum in the mid-IR region, even in the presence of up to a 30 μm thick water layer on top of a sample pattern. Additionally, the Q-IR technique offers practical advantages over synchrotron-based FTIR, such as reduced complexity, cost, and ease of operation.

The main proteases M pro are a group of highly conserved cysteine hydrolases in β-coronaviruses. They have been demonstrated to play an unavoidable role in viral replication, and consequently they have been suggested as key targets for treating coronavirus-caused infectious diseases, mainly from the COVID-19 epidemic. Since the most functional form for M pro enzymatic activity is associated to its homodimer, compounds inhibiting dimerization should also inhibit catalytic activity. We show how PIR-SEIRA (Plasmonic Internal Reflection-Surface Enhanced InfraRed Absorption) spectroscopy can be a noteworthy technique to study proteins subtle structural variations associated to inhibitor binding. Nanoantennas arrays can selectively confine and enhance electromagnetic field via localized plasmonic resonances, thus promoting ultrasensitive detection of biomolecules in close proximity of nanoantenna arrays and enabling the effective investigation of protein monolayers. By adopting this approach, reflection measurements conducted under back illumination of nanoantennas allow to probe anchored protein monolayers, with minimum contribution of environmental buffer molecules. PIR-SEIRA spectroscopy on M pro was carried out by ad hoc designed devices, resonating in the spectral region of Amide I and Amide II bands. We evaluated here the structure of anchored monomers and dimers in different buffered environment and in presence of a newly designed M pro inhibitor. Experimental results show that dimerization is not associated to relevant backbone rearrangements of the protein at secondary structure level, and even if the compound inhibits the dimerization, it is not effective at breaking preformed dimers., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Missense mutations in EGFR's catalytic domain alter its function, promoting cancer. SEIRA spectroscopy, supported by MD simulations, reveals structural differences in the compactness and hydration of helical motifs between active and inactive EGFR conformations models. These findings provide novel insights into the biophysical mechanisms driving EGFR activation and drug resistance, offering a robust method for studying emerging EGFR mutations and their structural impacts on TKIs' efficacy.

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.

Despite the extensive literature on the retrieval of digestible starches from archaeological contexts, there are still significant concerns regarding their genuine origin and durability. Here, we propose a multi-analytical strategy to identify the authenticity of ancient starches retrieved from macrolithic tools excavated at Upper Paleolithic sites in the Pontic steppe. This strategy integrates the morphological discrimination of starches through optical microscopy and scanning electron microscopy with single starch chemo-profiling using Fourier transform infrared imaging and microscopy. We obtained evidence of aging and biomineralization in the use-related starches from Palaeolithic sites, providing a methodology to establish their ancient origin, assess their preservation status, and attempt their identification. The pivotal application of this multidisciplinar approach demonstrates that the macrolithic tools, from which starches were dislodged, were used for food-processing across the Pontic Steppe around 40,000 years ago during the earliest colonization of Eurasia by Homo sapiens., (© 2023. The Author(s).)

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Zingaro, Gianoncelli, Ceccone, Birarda, Cassano, La Spina, Agostinis, Bonanni, Ricci and Pascolo.)

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.

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.

Coccolithophores, marine calcifying phytoplankton, are important primary producers impacting the global carbon cycle at different timescales. Their biomineral structures, the calcite containing coccoliths, are among the most elaborate hard parts of any organism. Understanding the morphogenesis of coccoliths is not only relevant in the context of coccolithophore eco-physiology but will also inform biomineralization and crystal design research more generally. The recent discovery of a silicon (Si) requirement for crystal shaping in some coccolithophores has opened up a new avenue of biomineralization research. In order to develop a mechanistic understanding of the role of Si, the presence and localization of this chemical element in coccoliths needs to be known. Here, we document for the first time the uneven Si distribution in Helicosphaera carteri coccoliths through three synchrotron-based techniques employing X-ray Fluorescence and Infrared Spectromicroscopy. The enrichment of Si in specific areas of the coccoliths point to a targeted role of this element in the coccolith formation. Our findings mark a key step in biomineralization research because it opens the door for a detailed mechanistic understanding of the role Si plays in shaping coccolith crystals., (© 2023. The Author(s).)

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 β-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 β-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., (© 2023. The Author(s).)

Antarctica has been affected directly and indirectly by human pressure for more than two centuries and recently plastic pollution has been recognized as a further potential threat for its unique biodiversity. Global long-range transport as well as local input from anthropogenic activities are potential sources of plastic pollution in both terrestrial and marine Antarctic territories. The present study evaluated the presence of microplastics in specimens of the Antarctic whelk Neobuccinum eatoni, a key species in benthic communities of the Ross Sea, one of the largest marine protected areas worldwide. To this aim, a thermo-oxidative extraction method was applied for microplastic isolation and quantification, and polymer identification was performed by manual μ-FTIR spectroscopy. Textile (semi-)synthetic or composite microfibers (length range: 0.8-5.7 mm) were found in 27.3% of whelk specimens, suggesting a low risk of bioaccumulation along Antarctic benthic food webs in the Ross Sea. Their polymer composition (of polyethylene terephthalate and cellulose-polyamide composites) matched those of outdoor technical clothing in use by the personnel of the Italian "Mario Zucchelli" station near Terra Nova Bay in the Ross Sea. Such findings indicate that sewage from base stations may act as potential local sources of textile microplastic fibers in this remote environment. More in-depth monitoring studies aiming at defining the extent of microplastic contamination related to such sources in Antarctica are encouraged., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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., (© 2022 the author(s), published by De Gruyter.)

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.

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., Competing Interests: There are no conflicts to declare., (This journal is © The Royal Society of Chemistry.)

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.

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 β-sheet conformation while parallel β-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.

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.

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

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.

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.

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 Si 3 N 4 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 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 (LC 50 -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., (Copyright © 2020. Published by Elsevier Ltd.)

In order to push the spatial resolution limits to the nanoscale, synchrotron-based soft X-ray microscopy (XRM) experiments require higher radiation doses to be delivered to materials. Nevertheless, the associated radiation damage impacts on the integrity of delicate biological samples. Herein, the extent of soft X-ray radiation damage in popular thin freeze-dried brain tissue samples mounted onto Si 3 N 4 membranes, as highlighted by Fourier transform infrared microscopy (FTIR), is reported. The freeze-dried tissue samples were found to be affected by general degradation of the vibrational architecture, though these effects were weaker than those observed in paraffin-embedded and hydrated systems reported in the literature. In addition, weak, reversible and specific features of the tissue-Si 3 N 4 interaction could be identified for the first time upon routine soft X-ray exposures, further highlighting the complex interplay between the biological sample, its preparation protocol and X-ray probe.

Plant material falling into the ultra-basic (pH 11.5-11.9) springs within The Cedars, an actively serpentinizing site in Sonoma County, California, is subject to conditions that mimic the industrial pretreatment of lignocellulosic biomass for biofuel production. We sought to obtain hemicellulolytic/cellulolytic bacteria from The Cedars springs that are capable of withstanding the extreme alkaline conditions wherein calcium hydroxide-rich water removes lignin, making cell wall polysaccharides more accessible to microorganisms and their enzymes. We enriched for such bacteria by adding plant debris from the springs into a synthetic alkaline medium with ground tissue of the biofuel crop switchgrass (Panicum virgatum L.) as the sole source of carbon. From the enrichment culture we isolated the facultative anaerobic bacterium Cellulomonas sp. strain FA1 (NBRC 114238), which tolerates high pH and catabolizes the major plant cell wall-associated polysaccharides cellulose, pectin, and hemicellulose. Strain FA1 in monoculture colonized the plant material and degraded switchgrass at a faster rate than the community from which it was derived. Cells of strain FA1 could be acclimated through subculturing to grow at a maximal concentration of 13.4% ethanol. A strain FA1-encoded β-1, 4-endoxylanase expressed in E. coli was active at a broad pH range, displaying near maximal activity at pH 6-9. Discovery of this bacterium illustrates the value of extreme alkaline springs in the search for microorganisms with potential for consolidated bioprocessing of plant biomass to biofuels and other valuable bio-inspired products.

There is evidence and serious concern that microplastics have reached the most remote regions of the planet, but how far have they travelled in terrestrial ecosystems? This study presents the first field-based evidence of plastic ingestion by a common and central component of Antarctic terrestrial food webs, the collembolan Cryptopygus antarcticus . A large piece of polystyrene (PS) foam (34 × 31 × 5 cm) covered by microalgae, moss, lichens and microfauna was found in a fellfield along the shores of the Fildes Peninsula (King George Island). The application of an improved enzymatic digestion coupled with Fourier transform infrared microscopy (µ-FTIR), unequivocally detected traces of PS (less than 100 µm) in the gut of the collembolans associated with the PS foam and documented their ability to ingest plastic. Plastics are thus entering the short Antarctic terrestrial food webs and represent a new potential stressor to polar ecosystems already facing climate change and increasing human activities. Future research should explore the effects of plastics on the composition, structure and functions of polar terrestrial biota.

Sediment-hosted CO 2 -rich aquifers deep below the Colorado Plateau (USA) contain a remarkable diversity of uncultivated microorganisms, including Candidate Phyla Radiation (CPR) bacteria that are putative symbionts unable to synthesize membrane lipids. The origin of organic carbon in these ecosystems is unknown and the source of CPR membrane lipids remains elusive. We collected cells from deep groundwater brought to the surface by eruptions of Crystal Geyser, sequenced the community, and analyzed the whole community lipidome over time. Characteristic stable carbon isotopic compositions of microbial lipids suggest that bacterial and archaeal CO 2 fixation ongoing in the deep subsurface provides organic carbon for the complex communities that reside there. Coupled lipidomic-metagenomic analysis indicates that CPR bacteria lack complete lipid biosynthesis pathways but still possess regular lipid membranes. These lipids may therefore originate from other community members, which also adapt to high in situ pressure by increasing fatty acid unsaturation. An unusually high abundance of lysolipids attributed to CPR bacteria may represent an adaptation to membrane curvature stress induced by their small cell sizes. Our findings provide new insights into the carbon cycle in the deep subsurface and suggest the redistribution of lipids into putative symbionts within this community.

The Krapina white-tailed eagle talons represent a kind of jewelry worn by Krapina Neandertals some 130,000 years ago. New inspection of one Krapina talon (386.1) revealed a fiber, sealed by a thin silicate coating, adhering to the surface within a wide cut mark, as well as concentrated traces of occasional spots of red and yellow pigment and some black stains. We analyzed the fiber and small portions of pigmented areas by non-invasive, infrared synchrotron beam. Different areas were targeted, revealing the protein nature of the fiber, identified as of animal origin. Targeted areas revealed intra- and inter-strand aggregation indicating the fiber to be collagen losing its original triple α-helix conformation, further confirming the diagenetic decay of the original collagen structure and the antiquity of the fiber. It is possible that the fiber is a remnant of the leather or sinew string binding the talons together. Spectroscopic analysis of the pigments in two isolated areas confirmed two types of ochre and that the dark spots are charcoal remnants. Applying novel non-invasive technologies provides new possibilities to further test the hypothesis of using prehistoric objects for symbolic purposes.

Model-based algorithms have recently attracted much attention for data pre-processing in tissue mapping and imaging by Fourier transform infrared micro-spectroscopy (FTIR). Their versatility, robustness and computational performance enabled the improvement of spectral quality by mitigating the impact of scattering and fringing in FTIR spectra of chemically homogeneous biological systems. However, to date, no comprehensive algorithm has been optimized and automated for large-area FTIR imaging of histologically complex tissue samples. Herein, for the first time, we propose a unique, integrated and fully-automated Multiple Linear Regression Multi-Reference (MLR-MR) method for correcting linear baseline effects due to diffuse scattering, for compensating substrate thickness inhomogeneity and accounting for sample chemical heterogeneity in FTIR images. In particular, the algorithm uses multiple-reference spectra for histologically heterogeneous biological samples. The performance of the procedure was demonstrated for FTIR imaging of chemically complex rat brain frontal cortex tissue samples, mounted onto Ultralene® films. The proposed MLR-MR correction algorithm allows the efficient retrieval of "pure" absorbance spectra and greatly improves the histological fidelity of FTIR imaging data, as compared with the one-reference approach. In addition, the MLR-MR algorithm here presented opens up the possibility for extracting information on substrate thickness variability, thus enabling the indirect evaluation of its topography. As a whole, the MLR-MR procedure can be easily extended to more complex systems for which Mie scattering effects must also be eliminated., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2019 Elsevier B.V. All rights reserved.)

This work presents a thorough study on the Amide III band in fibrous proteins using Fourier Transformed Infrared Spectroscopy (FTIR). Type I collagen was chosen as a model for this family of proteins, not only because of its important role in mammalian tissues, but also for its involvement in several pathologies. In order to disclose the conformational information contained in the collagen bands, the spectral characteristics of Amide III of type I collagen were related to the ones of Amide I band, performing experiments of thermal denaturation of the protein in acidic solution. Data acquired allowed to observe the protein unfolding and retrieve information about its structural arrangements during the thermal cycle. Taking as guideline the well-known behaviour of the Amide I band, we correlated the structural changes deducible from Amide I analysis with the ones detectable for Amide III band, by exploiting three spectral analysis techniques, namely 2D-correlation analysis, second derivative analysis, and peak-fitting. This approach enabled us to jointly support the obtained results and finally to assign the components of the Amide III of a typical fibrous protein, such as type I collagen, to its characteristic secondary structure., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Polyfurfuryl alcohol (PFA) is one of the most intriguing polymers because, despite its easy polymerization in acid environment, its molecular structure is definitely not obvious. Many studies have been performed in recent decades, and every time, surprising aspects came out. With the present study, we aim to take advantage of all of the findings of previous investigations and exploit them for the interpretation of the completely cured PFA spectra registered with three of the most powerful techniques for the characterization of solid, insoluble polymers: Solid-State 13 C-NMR, Attenuated Total Reflectance (ATR), Fourier Transform Infrared (FTIR) spectroscopy, and UV-resonant Raman spectroscopy at different excitation wavelengths, using both an UV laser source and UV synchrotron radiation. In addition, the foreseen structures were modeled and the corresponding 13 C-NMR and FTIR spectra were simulated with first-principles and semi-empiric methods to evaluate their matching with experimental ones. Thanks to this multi-technique approach, based on complementary analytical tools and computational support, it was possible to conclude that, in addition to the major linear unconjugated polymerization, the PFA structure consists of Diels-Alder rearrangements occurring after the opening of some furanic units, while the terminal moieties of the chain involves γ-lactone arrangements. The occurrence of head-head methylene ether bridges and free hydroxyl groups (from unreacted furfuryl alcohol, FA, or terminal chains) could be excluded, while the conjugated systems could be considered rather limited.

Biofilms are communities of bacteria living embedded in a highly hydrated matrix composed of polysaccharides, proteins, and extracellular DNA. This life style confers numerous advantages to bacteria including protection against external threats. However, they also contribute to increase bacterial resistance against antimicrobials, an issue particularly relevant in dangerous infections. Due to the complexity of the matrix, few information is present in the literature on details of its architecture including the spatial distribution of the macromolecular components which might give hints on the way the biofilm scaffold is built up by bacteria. In this study, we investigated the possibility to combine well-established microbiological procedures with advanced microscopies to get information on composition and distribution of the macromolecular components of biofilm matrices. To this, confocal microscopy, diffraction-limited infrared (IR) spectral imaging, and atomic force microscopy (AFM) were used to explore biofilm produced by a clinical strain of Klebsiella pneumoniae. IR imaging permitted to have clues on how the biofilm grows and spreads on surfaces, and the local distribution of the components within it. Through the analysis of the pure component spectra, it was possible to assess the chemical and structural composition of the saccaridic matrix, confirming the data obtained by NMR. It was also possible to follow the time course of biofilm from 6 up to 48 h when the biofilm grew into a 3-dimensional multi-layered structure, characteristic of colonies of bacteria linked together by a complex matrix. In addition, nanoFTIR and AFM investigations allowed the estimation of biofilm growth in the vertical direction and the morphological analysis of bacterial colonies at different time points and the evaluation of the chemical composition at the nanoscale.

Microscopic analysis of backed lithic pieces from the Uluzzian technocomplex (45-40 thousand yr ago) at Grotta del Cavallo (southern Italy) reveals their use as mechanically delivered projectile weapons, attributed to anatomically modern humans. Use-wear and residue analyses indicate that the lithics were hunting armatures hafted with complex adhesives, while experimental and ethnographic comparisons support their use as projectiles. The use of projectiles conferred a hunting strategy with a higher impact energy and a potential subsistence advantage over other populations and species.

Juncus acutus, an halophite plant pioneer in extremely polluted mine areas, was harvested in three different locations of Sardinia (Italy), having Zn soil concentration up to 80g/kg, and Zn water concentration ranging between 10 -3 g/L and 10 -1 g/L. Rhizosphere and plant samples were investigated combining X-ray microscopy (XM)/spectroscopy (XAFS) and infrared microspectroscopy (FTIR) to elucidate the chemical composition, (bio)mineralogy and Zn coordinative environment. The multi-technique approach allowed recognizing different biomineralization processes, and Zn complexes in the plant tissues. The Zn chemical environment in root biominerals is multi-phase and, depending on the sampling site, can comprise amorphous Zn silicate, Zn apatite, hydrozincite, and Zn sulphate. Zn cysteine and Zn histidine, complexes quoted as part of a detoxification strategy, were found mainly in plants from the site where the Zn water concentration has the highest values. This different site-specific mode of Zn biomineralization has relevant implications for phytoremediation techniques and for further biotechnology development, which can be better designed and developed after knowledge of site-specific-molecular processes ruling mineral evolution and biomineralization. Carboxylic groups and organic compounds (lignin, cellulose, hemicellulose, pectin and esters) were identified by FTIR analysis, thought the Zn speciation is not apparently linked to these carboxylic group rich biopolymers., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: Technical limitations regarding bulk analysis of phytoplankton biomass limit our comprehension of carbon fluxes in natural populations and, therefore, of carbon, nutrients and energy cycling in aquatic ecosystems. In this study, we took advantage of Synchrotron FTIR micro-spectroscopy and the partial least square regression (PLSr) algorithm to simultaneously quantify the protein, lipid and carbohydrate content at the single-cell level in a mock phytoplankton community (composed by a cyanobacterium, a green-alga and a diatom) grown at two temperatures (15 °C and 25 °C)., Results: The PLSr models generated to quantify cell macromolecules presented high quality fit (R 2  ≥ 0.90) and low error of prediction (RMSEP 2-6% of dry weight). The regression coefficients revealed that the prediction of each macromolecule was not exclusively dependent on spectral features corresponding to that compound, but rather on all major macromolecular pools, reflecting adjustments in the overall cell carbon balance. The single-cell analysis, studied by means of Kernel density estimators, showed that the modes of density distribution of macromolecules were different at 15 °C and 25 °C. However, a substantial proportion of cells was biochemically identical at the two temperatures because of population heterogeneity., Conclusions: The spectroscopic approach presented in this study allows the quantification of macromolecules in single phytoplankton cells. This method showed that population heterogeneity most likely ensures a backup of non-acclimated cells that may rapidly exploit new favourable niches. This finding may have important consequences for the ecology of phytoplankton populations and shows that the "average cell" concept might substantially limit our comprehension of population dynamics and biogeochemical cycles in aquatic ecosystems.

The present work aims to study the effects that acute exposure to low concentrations of silver nanoparticles (AgNPs) cause in digestive glands of terrestrial isopods (Porcellio scaber). The experiments were designed to integrate different analytical techniques, such as transmission electron microscopy, atomic absorption spectroscopy, proton induced X-ray emission, and Fourier transform IR imaging (FTIRI), in order to gain a comprehensive insight into the process from the AgNPs' synthesis to their interaction with biological tissues in vivo. To this aim, terrestrial isopods were fed with AgNPs having different shapes, sizes, and concentrations. For all the tested conditions, no toxicity at the whole organism level was observed after 14 days of exposure. However, FTIRI showed that AgNPs caused detectable local changes in proteins, lipids, nucleic acids and carbohydrates at the tissue level, to an extent dependent on the interplay of the AgNPs' properties: shape, size, concentration and dissolution of ions from them.