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Kaposi sarcoma herpesvirus/human herpesvirus-8 (HHV-8) neoplastic and non-neoplastic disease in solid organ transplant (SOT) recipients can be life-threatening. We evaluated the seroprevalence of HHV-8 infection among donors (D) and recipients (R), the incidence of HHV-8 transmission/reactivation, and the clinical characteristics, management, and outcomes of HHV-8-related diseases, including Kaposi sarcoma herpesvirus-associated inflammatory cytokine syndrome (KICS), in consecutive SOT patients from 2011 to 2023. HHV-8 seroprevalence was 3.3% in 1349 donors and 8.4% in 1856 recipients screened (p<0.0001). Among the D+/R- group (n=49), 13 patients developed HHV-8-related diseases: 7 liver recipients had KICS, and 1 lung recipient had Kaposi sarcoma (KS) with subsequent KICS. Four KICS patients treated with rituximab survived, while the 3 patients not treated with rituximab died. Within the D-/R- group, out of 5 (0.3%) patients with non-donor-derived primary HHV-8 infection, 3 liver recipients developed KICS. In the R+ patients (n=155), 3 developed KS. In our cohort, 25/944 (2.6%) liver transplant recipients had a primary HHV-8 infection, and 10 of them (40%) developed KICS; 40% (4/10) of HHV-8 seropositive heart transplant recipients developed reactivation and 2 of them (50%) had fatal KS. Serologic screening and molecular surveillance of D+/R- patient groups facilitate early recognition and effective therapy of KICS., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Shifts in cytosolic pH have been recognized as key signaling events and mounting evidence supports the interdependence between H + and Ca 2+ signaling in eukaryotic cells. Among the cellular pH-stats, K + /H + exchange at various membranes is paramount in plant cells. Vacuolar K + /H + exchangers of the NHX (Na + ,K + /H + exchanger) family control luminal pH and, together with K + and H + transporters at the plasma membrane, have been suggested to also regulate cytoplasmic pH. We show the regulation of vacuolar K + /H + exchange by cytoplasmic pH and the calmodulin-like protein CML18 in Arabidopsis. The crystal structure and physicochemical properties of CML18 indicate that this protein senses pH shifts. Interaction of CML18 with tonoplast exchangers NHX1 and NHX2 was favored at acidic pH, a physiological condition elicited by K + starvation in Arabidopsis roots, whereas excess K + produced cytoplasmic alkalinization and CML18 dissociation. These results imply that the pH-responsive NHX-CML18 module is an essential component of the cellular K + - and pH-stats.

In recent decades, driven by the needs of industry and medicine, researchers have been investigating how to remove carefully from the main flow microscopic particles or clusters of them. Among all the approaches proposed, crossflow filtration is one of the most attractive as it provides a non-destructive, label-free and in-flow sorting method. In general, the separation performance shows capture and separation efficiencies ranging from 70% up to 100%. However, the maximum flow rate achievable (µL/min) is still orders of magnitude away from those suitable for clinical or industrial applications mainly due to the low stiffness of the materials typically used. In this work, we propose an innovative hydrodynamic-crossflow hybrid filter geometry, buried in a fused silica substrate by means of the femtosecond laser irradiation followed by chemical etching technique. The material high stiffness combined with the accuracy of our manufacturing technique allows the 3D fabrication of non-deformable channels with higher aspect ratio posts, while keeping the overall device dimensions compact. The filter performance has been validated through experiments with both Newtonian (water-based solution of microbeads) and non-Newtonian fluids (blood), achieving separation efficiencies of up to 94% and large particles recovery rates of 100%, even at very high flow rates (mL/h)., (© 2024 Wiley‐VCH GmbH.)

Despite extensive research efforts, cancer continues to stand as one of the leading causes of death on a global scale. To gain profound insights into the intricate mechanisms underlying cancer onset and progression, it is imperative to possess methodologies that allow the study of cancer cells at the single-cell level, focusing on critical parameters such as cell morphology, metabolism, and molecular characteristics. These insights are essential for effectively discerning between healthy and cancerous cells and comprehending tumoral progression. Recent advancements in microscopy techniques have significantly advanced the study of cancer cells, with Raman microspectroscopy (RM) emerging as a particularly powerful tool. Indeed, RM can provide both biochemical and spatial details at the single-cell level without the need for labels or causing disruptions to cell integrity. Moreover, RM can be correlated with other microscopy techniques, creating a synergy that offers a spectrum of complementary insights into cancer cell morphology and biology. This review aims to explore the correlation between RM and other microscopy techniques such as confocal fluoresce microscopy (CFM), atomic force microscopy (AFM), digital holography microscopy (DHM), and mass spectrometry imaging (MSI). Each of these techniques has their own strengths, providing different perspectives and parameters about cancer cell features. The correlation between information from these various analysis methods is a valuable tool for physicians and researchers, aiding in the comprehension of cancer cell morphology and biology, unraveling mechanisms underlying cancer progression, and facilitating the development of early diagnosis and/or monitoring cancer progression.

Background: Breast cancer (BC) is a heterogeneous neoplasm characterized by several subtypes. One of the most aggressive with high metastasis rates presents overexpression of the human epidermal growth factor receptor 2 (HER2). A quantitative evaluation of HER2 levels is essential for a correct diagnosis, selection of the most appropriate therapeutic strategy and monitoring the response to therapy., Results: In this paper, we propose the synergistic use of SERS and Raman technologies for the identification of HER2 expressing cells and its accurate assessment. To this end, we selected SKBR3 and MDA-MB-468 breast cancer cell lines, which have the highest and lowest HER2 expression, respectively, and MCF10A, a non-tumorigenic cell line from normal breast epithelium for comparison. The combined approach provides a quantitative estimate of HER2 expression and visualization of its distribution on the membrane at single cell level, clearly identifying cancer cells. Moreover, it provides a more comprehensive picture of the investigated cells disclosing a metabolic signature represented by an elevated content of proteins and aromatic amino acids. We further support these data by silencing the HER2 gene in SKBR3 cells, using the RNA interference technology, generating stable clones further analysed with the same combined methodology. Significant changes in HER2 expression are detected at single cell level before and after HER2 silencing and the HER2 status correlates with variations of fatty acids and downstream signalling molecule contents in the context of the general metabolic rewiring occurring in cancer cells. Specifically, HER2 silencing does reduce the growth ability but not the lipid metabolism that, instead, increases, suggesting that higher fatty acids biosynthesis and metabolism can occur independently of the proliferating potential tied to HER2 overexpression., Conclusions: Our results clearly demonstrate the efficacy of the combined SERS and Raman approach to definitely pose a correct diagnosis, further supported by the data obtained by the HER2 gene silencing. Furthermore, they pave the way to a new approach to monitor the efficacy of pharmacologic treatments with the aim to tailor personalized therapies and optimize patients' outcome., (© 2024. The Author(s).)

To control net sodium (Na + ) uptake, Arabidopsis plants utilize the plasma membrane (PM) Na + /H + antiporter SOS1 to achieve Na + efflux at the root and Na + loading into the xylem, and the channel-like HKT1;1 protein that mediates the reverse flux of Na + unloading off the xylem. Together, these opposing transport systems govern the partition of Na + within the plant yet they must be finely co-regulated to prevent a futile cycle of xylem loading and unloading. Here, we show that the Arabidopsis SOS3 protein acts as the molecular switch governing these Na + fluxes by favoring the recruitment of SOS1 to the PM and its subsequent activation by the SOS2/SOS3 kinase complex under salt stress, while commanding HKT1;1 protein degradation upon acute sodic stress. SOS3 achieves this role by direct and SOS2-independent binding to previously unrecognized functional domains of SOS1 and HKT1;1. These results indicate that roots first retain moderate amounts of salts to facilitate osmoregulation, yet when sodicity exceeds a set point, SOS3-dependent HKT1;1 degradation switches the balance toward Na + export out of the root. Thus, SOS3 functionally links and co-regulates the two major Na + transport systems operating in vascular plants controlling plant tolerance to salinity., Competing Interests: Competing interests statement:The authors declare no competing interest.

The actin filaments on the surface of echinoderm oocytes and eggs readily undergo massive reorganization during meiotic maturation and fertilization. In sea urchin eggs, the actin cytoskeletal response to the fertilizing sperm is fast enough to accompany Ca 2+ signals and to guide sperm's entry into the egg. Although recent work using live cell imaging technology confirmed changes in the actin polymerization status in fertilized eggs, as was previously shown using light and electron microscopy, it failed to provide experimental evidence of F-actin depolymerization a few seconds after insemination, which is concurrent with the sperm-induced Ca 2+ release. In the present study, we applied Raman microspectroscopy to tackle this issue by examining the spectral profiles of the egg's subplasmalemmal regions before and after treating the eggs with actin drugs or fertilizing sperm. At both early (15 s) and late (15 min) time points after fertilization, specific peak shifts in the Raman spectra revealed change in the actin structure, and Raman imaging detected the cytoskeletal changes corresponding to the F-actin reorganization visualized with LifeAct-GFP in confocal microscopy. Our observation suggests that the application of Raman spectroscopy, which does not require microinjection of fluorescent probes and exogenous gene expression, may serve as an alternative or even advantageous method in disclosing rapid subtle changes in the subplasmalemmal actin cytoskeleton that are difficult to resolve.

Sulfur dioxide (SO 2 ) and sulfites are well-known additives in winemaking due to their preservative properties. Although they can prevent oxidation and inhibit microbial growth, they pose health risks and require limitations on their use. Consequently, the total level of SO 2 is regulated and several quantification strategies have been proposed. The approved detection methods require the extraction of SO 2 by heating and/or acid treatment. Then, iodine or acid/base titrations are conducted for the detection of liberated SO 2 . Although these methods can provide sensitive detection of SO 2 , they are complex, time-consuming, and require sample preparation steps and skilled operators. Thus, to overcome these disadvantages, an easy-to-use method, involving simple sample preparation steps, and offering high sensitivity and selectivity, is desirable. Herein, we introduce a SERS-based strategy for SO 2 detection in liquids using hydrogel nanocomposites. The hydrogels are prepared by poly(ethylene glycol) diacrylate (PEGDA) in the presence of gold nanoparticles (AuNPs), acting as the SERS substrate. The use of hydrogels ensures a homogenous signal distribution and an efficient collection of SO 2 , and drying the hydrogels enhances and stabilizes the obtained SO 2 signal. The detection strategy is based on the pH-dependent dissociation of SO 2 . By adjusting the pH value of wine to 10 through simple dilutions, SO 2 can be directly detected in wine, down to 0.4 ppm, well below the regulatory limits. The proposed method allows for sensitive, direct, cost-effective detection of SO 2 by eliminating the loss of the gaseous form of the sample and avoids titration-based detection methods., 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 © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Aromatase inhibitors (AIs) show promising features as drugs to treat estrogen-responsive breast cancer as they block aromatase activity, the key enzyme in estrogen synthesis. The current AIs approved by the Food and Drug Administration for breast cancer treatment present severe adverse effects. For these reasons, it is important to develop of new AIs that are more specific and sensitive. In this paper, we report the synthesis and the characterization of new nonsteroidal aromatase AIs containing triazoles moieties for the treatment of hormone-dependent breast cancer in post-menopausal women. A new series of 1,2,3-triazole based molecules were successfully synthetized and their chemical structures were determined from the spectral data (FT-IR, 13 C NMR, 1 H NMR, mass spectroscopy) and micro-analytical data. Additionally, the physical properties of the newly synthesized derivatives were reported. The novel compounds were also tested for their anticancer activity in both breast cancer (MCF7 and T-47D) and normal breast (MCF 10A) cell lines, evaluating their effect on cell proliferation, migration, and invasion. The results revealed that the compounds exhibited promising and specific anti-cancer action.

Circulating tumor cells (CTCs) are tumor cells that have penetrated the circulatory system preserving tumor properties and heterogeneity. Detection and characterization of CTCs has high potential clinical values and many technologies have been developed for CTC identification. These approaches remain challenged by the extraordinary rarity of CTCs and the difficulty of efficiently distinguishing cancer from the much larger number of white blood cells in the bloodstream. Consequently, there is still a need for efficient and rapid methods to capture the broad spectrum of tumor cells circulating in the blood. Herein, we exploit the peculiarities of cancer metabolism for discriminating cancer from WBCs. Using deuterated glucose and Raman microscopy we show that a) the known ability of cancer cells to take up glucose at greatly increased rates compared to non-cancer cells results in the lipid generation and accumulation into lipid droplets and, b) by contrast, leukocytes do not appear to generate visible LDs. The difference in LD abundance is such that it provides a reliable parameter for distinguishing cancer from blood cells. For LD sensitive detections in a cell at rates suitable for screening purposes, we test a polarization-sensitive digital holographic imaging (PSDHI) technique that detects the birefringent properties of the LDs. By using polarization-sensitive digital holographic imaging, cancer cells (prostate cancer, PC3 and hepatocarcinoma cells, HepG2) can be rapidly discriminated from leukocytes with reliability close to 100%. The combined Raman and PSDHI microscopy platform lays the foundations for the future development of a new label-free, simple and universally applicable cancer cells' isolation method., 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 Mangini, Ferrara, Zito, Managò, Luini, De Luca and Coppola.)

Profilin 1-encoded by PFN1-is a small actin-binding protein with a tumour suppressive role in various adenocarcinomas and pagetic osteosarcomas. However, its contribution to tumour development is not fully understood. Using fix and live cell imaging, we report that Profilin 1 inactivation results in multiple mitotic defects, manifested prominently by anaphase bridges, multipolar spindles, misaligned and lagging chromosomes, and cytokinesis failures. Accordingly, next-generation sequencing technologies highlighted that Profilin 1 knock-out cells display extensive copy-number alterations, which are associated with complex genome rearrangements and chromothripsis events in primary pagetic osteosarcomas with Profilin 1 inactivation. Mechanistically, we show that Profilin 1 is recruited to the spindle midzone at anaphase, and its deficiency reduces the supply of actin filaments to the cleavage furrow during cytokinesis. The mitotic defects are also observed in mouse embryonic fibroblasts and mesenchymal cells deriving from a newly generated knock-in mouse model harbouring a Pfn1 loss-of-function mutation. Furthermore, nuclear atypia is also detected in histological sections of mutant femurs. Thus, our results indicate that Profilin 1 has a role in regulating cell division, and its inactivation triggers mitotic defects, one of the major mechanisms through which tumour cells acquire chromosomal instability., (© 2023. The Author(s).)

Circular economy (CE) is claimed to be a promising pathway to achieve the Sustainable Development Goals (SDGs), but a reliable metric is needed to validate closed-loop strategies by measuring sustainability performances together with the degree of circularity. A significant contribution is offered by Life Cycle (LC) scholars in terms of methodological advances and operational tools for different sectors, also those more complex such as the agro-industrial systems that encompass biological and anthropogenic variables at different scales. However, to date, LC methodologies have not yet answered how to model the complexity of circular pathways. LC evaluations are often modelled for cradle-to-grave analyses, while a circularity evaluation would require an extension of the system boundaries to more interconnected life cycles, orienting towards a cradle-to-cradle perspective. This research gap led us to propose a multi-cycle approach with expanded assessment boundaries, including co-products, into a cradle-to-cradle perspective, in an attempt to internalize circularity impacts. The customized LC framework here proposed is based on the Life Cycle Assessment (LCA), the Environmental Life Cycle Costing (ELCC) in terms of internal and external costs, and the Social Life Cycle Assessment (SLCA) in terms of Psychosocial Risk Factor (PRF) impact pathway. The model is designed to be applied to the olive-oil sector, which commonly causes significant impacts by generating many by-products whose management is often problematic. Results are expected to show that the customized LC framework proposed can better highlight the environmental and socioeconomic performances of the system of cycles, allowing CE to deliver its promises of sustainability, as the circularity of materials per se is a means, not an end in itself., 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 B.V. All rights reserved.)

Several living organisms are able to synthesize complex nanostructures provided with peculiar physical and chemical properties by means of finely-tuned, genetically controlled biomineralization processes. Frustules, in particular, are micro- and nano-structured silica shells produced by ubiquitous diatom microalgae, whose optical properties have been recently exploited in photonics, solar energy harvesting, and biosensing. Metallization of diatom biosilica, both in the shape of intact frustules or diatomite particles, can trigger plasmonic effects that in turn can find application in high-sensitive detection platforms, allowing to obtain effective nanosensors at low cost and on a large scale. The aim of the present review article is to provide a wide, complete overview on the main metallization techniques applied to diatom biosilica and on the principal applications of diatom-based plasmonic devices mainly but not exclusively in the fields of biochemical sensing, diagnostics and therapeutics., Competing Interests: The authors declare no conflicts of interest., (© 2022 Optica Publishing Group under the terms of the Optica Open Access Publishing Agreement.)

The prevalence of patients affected by end-stage diseases or advanced cancer is increasing due to an aging population and progression in medicine and public healthcare. The burden of symptoms these people suffer in the last months of life often forces them to seek aid in an emergency department. In developed countries, acute care hospital-based services are often better designed to treat acute clinical conditions than to manage the needs of patients with serious chronic diseases. Thus, the palliative care (PC) population poses very real clinical challenges to healthcare professionals who care for them in hospital settings. The authors have formulated four key questions (who, why, when, and how) to address in order to identify a model for providing the best care for these PC patients. The questions are related to: (1) defining people living with serious chronic diseases; (2) managing the challenge of unplanned hospital admission of these people; (3) identifying PC patients among people with serious chronic diseases; and (4) determining the appropriate work of caring for this inpatient PC population. Clinicians need the knowledge, tools, and services to care for these PC patients, and acute care hospitals should plan the work of caring for these inpatients.

Innate immune memory, the ability of innate cells to react in a more protective way to secondary challenges, is induced by exposure to infectious and other exogeous and endogenous agents. Engineered nanoparticles are particulate exogenous agents that, as such, could trigger an inflammatory reaction in monocytes and macrophages and could therefore be also able to induce innate memory. Here, we have evaluated the capacity of engineered gold nanoparticles (AuNPs) to induce a memory response or to modulate the memory responses induced by microbial agents. Microbial agents used were in soluble vs . particulate form (MDP and the gram-positive bacteria Staphylococcus aureus ; β-glucan and the β-glucan-producing fungi C. albicans ), and as whole microrganisms that were either killed ( S. aureus , C. albicans ) or viable (the gram-negative bacteria Helicobacter pylori ). The memory response was assessed in vitro , by exposing human primary monocytes from 2-7 individual donors to microbial agents with or without AuNPs (primary response), then resting them for 6 days to allow return to baseline, and eventually challenging them with LPS (secondary memory response). Primary and memory responses were tested as production of the innate/inflammatory cytokine TNFα and other inflammatory and anti-inflammatory factors. While inactive on the response induced by soluble microbial stimuli (muramyl dipeptide -MDP-, β-glucan), AuNPs partially reduced the primary response induced by whole microorganisms. AuNPs were also unable to directly induce a memory response but could modulate stimulus-induced memory in a circumscribed fashion, limited to some agents and some cytokines. Thus, the MDP-induced tolerance in terms of TNFα production was further exacerbated by co-priming with AuNPs, resulting in a less inflammatory memory response. Conversely, the H. pylori -induced tolerance was downregulated by AuNPs only relative to the anti-inflammatory cytokine IL-10, which would lead to an overall more inflammatory memory response. These effects of AuNPs may depend on a differential interaction/association between the reactive particle surfaces and the microbial components and agents, which may lead to a change in the exposure profiles. As a general observation, however, the donor-to-donor variability in memory response profiles and reactivity to AuNPs was substantial, suggesting that innate memory depends on the individual history of exposures., 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 © 2021 Swartzwelter, Michelini, Frauenlob, Barbero, Verde, De Luca, Puntes, Duschl, Horejs-Hoeck, Italiani and Boraschi.)

Surface-enhanced Raman spectroscopy (SERS) has become a powerful tool for biosensing applications owing to its fingerprint recognition, high sensitivity, multiplex detection, and biocompatibility. This review provides an overview of the most significant aspects of SERS for biomedical and biosensing applications. We first introduced the mechanisms at the basis of the SERS amplifications: electromagnetic and chemical enhancement. We then illustrated several types of substrates and fabrication methods, with a focus on gold-based nanostructures. We further analyzed the relevant factors for the characterization of the SERS sensor performances, including sensitivity, reproducibility, stability, sensor configuration (direct or indirect), and nanotoxicity. Finally, a representative selection of applications in the biomedical field is provided.

Engineered gold nanoparticles (AuNPs) find application in several fields related to human activities ( i.e. , food and cosmetic industry or water purification) including medicine, where they are employed for diagnosis, drug delivery and cancer therapy. As for any material/reagent for human use, the safety of AuNPs needs accurate evaluation. AuNPs are prone to contamination by bacterial endotoxin (lipopolysaccharide, LPS), a potent elicitor of inflammatory responses in mammals. It is therefore important, when assessing AuNP immunosafety and immune-related effects, to discriminate between inflammatory effects intrinsic to the NPs from those caused by an undeliberate and undetected LPS contamination. Detection of LPS contamination in AuNP preparations poses different problems when using the current LPS detection assays, given the general interference of NPs, similar to other particulate agents, with the assay reagents and endpoints. This leads to time-consuming search for optimal assay conditions for every NP batch, with unpredictable results, and to the use in parallel of different assays, each with its weaknesses and unpredictability. Thus, the development of highly sensitive, quantitative and accurate assays able to detect of LPS on AuNPs is very important, in view of their medical applications. Surface-enhanced Raman spectroscopy (SERS) is a label-free, sensitive, chemical-specific, nondestructive and fast technique that can be used to directly obtain molecular fingerprint information and a quantitative analysis of LPS adsorbed on AuNPs. Within this study, we describe the use of SERS for the label-free identification and quantitative evaluation - down to few attograms - of the LPS adsorbed on the surface of 50 nm AuNPs. We thus propose SERS as an efficient tool to detect LPS on the AuNP surface, and as the basis for the development of a new sensitive and specific LPS-detection sensor based on the use of AuNPs and SERS., 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 © 2021 Verde, Mangini, Managò, Tramontano, Rea, Boraschi, Italiani and De Luca.)

The Salt-Overly-Sensitive (SOS) pathway controls the net uptake of sodium by roots and the xylematic transfer to shoots in vascular plants. SOS3/CBL4 is a core component of the SOS pathway that senses calcium signaling of salinity stress to activate and recruit the protein kinase SOS2/CIPK24 to the plasma membrane to trigger sodium efflux by the Na/H exchanger SOS1/NHX7. However, despite the well-established function of SOS3 at the plasma membrane, SOS3 displays a nucleo-cytoplasmic distribution whose physiological meaning is not understood. Here, we show that the N-terminal part of SOS3 encodes structural information for dual acylation with myristic and palmitic fatty acids, each of which commands a different location and function of SOS3. N -myristoylation at glycine-2 is essential for plasma membrane association and recruiting SOS2 to activate SOS1, whereas S -acylation at cysteine-3 redirects SOS3 toward the nucleus. Moreover, a poly-lysine track in positions 7-11 that is unique to SOS3 among other Arabidopsis CBLs appears to be essential for the correct positioning of the SOS2-SOS3 complex at the plasma membrane for the activation of SOS1. The nuclear-localized SOS3 protein had limited bearing on the salt tolerance of Arabidopsis . These results are evidence of a novel S -acylation dependent nuclear trafficking mechanism that contrasts with alternative subcellular targeting of other CBLs by S -acylation., 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 © 2021 Villalta, García, Hornero-Mendez, Carranco, Tello, Mendoza, De Luca, Andrés, Schumacher, Pardo and Quintero.)

The small molecule Galunisertib (LY2157299, LY) shows multiple anticancer activities blocking the transforming growth factor-β1 receptor, responsible for the epithelial-to-mesenchymal transition (EMT) by which colorectal cancer (CRC) cells acquire migratory and metastatic capacities. However, frequent dosing of LY can produce highly toxic metabolites. Alternative strategies to reduce drug side effects can rely on nanoscale drug delivery systems that have led to a medical revolution in the treatment of cancer, improving drug efficacy and lowering drug toxicity. Here, a hybrid nanosystem (DNP-AuNPs-LY@Gel) made of a porous diatomite nanoparticle decorated with plasmonic gold nanoparticles, in which LY is retained by a gelatin shell, is proposed. The multifunctional capability of the nanosystem is demonstrated by investigating the efficient LY delivery, the enhanced EMT reversion in CRCs and the intracellular quantification of drug release with a sub-femtogram resolution by surface-enhanced Raman spectroscopy (SERS). The LY release trigger is the pH sensitivity of the gelatin shell to the CRC acidic microenvironment. The drug release is real-time monitored at single-cell level by analyzing the SERS signals of LY in CRC cells. The higher efficiency of LY delivered by the DNP-AuNPs-LY@Gel complex paves the way to an alternative strategy for lowering drug dosing and consequent side effects., (© 2021 The Authors. Small published by Wiley-VCH GmbH.)

Anaerobic codigestion of olive mill wastewater for renewable energy production constitutes a promising process to overcome management and environmental issues due to their conventional disposal. The present study aims at assessing biogas and biomethane production from olive mill wastewater by performing biochemical methane potential tests. Hence, mixtures containing 0% (blank), 20% and 30% olive mill wastewater, in volume, were experimented on under mesophilic conditions. In addition, life cycle assessment and life cycle costing were performed for sustainability analysis. Particularly, life cycle assessment allowed assessing the potential environmental impact resulting from the tested process, while life cycle costing in conjunction with specific economic indicators allowed performing the economic feasibility analysis. The research highlighted reliable outcomes: higher amounts of biogas (80.22 ± 24.49 NL.kg SV -1 ) and methane (47.68 ± 17.55 NL.kg SV -1 ) were obtained when implementing a higher amount of olive mill wastewater (30%) ( v / v ) in the batch reactors. According to life cycle assessment, the biogas ecoprofile was better when using 20% ( v / v ) olive mill wastewater. Similarly, the economic results demonstrated the profitability of the process, with better performances when using 20% ( v / v ) olive mill wastewater. These findings confirm the advantages from using farm and food industry by-products for the production of renewable energy as well as organic fertilizers, which could be used in situ to enhance farm sustainability.

The interaction between engineered nanoparticles and the bacterial lipopolysaccharide, or endotoxin, is an event that warrants attention. Endotoxin is one of the most potent stimulators of inflammation and immune reactions in human beings, and is a very common contaminant in research labs. In nanotoxicology and nanomedicine, the presence of endotoxin on the nanoparticle surface affects their biological properties leading to misinterpretation of results. This review discusses the importance of detecting the endotoxin contamination on nanoparticles, focusing on the current method of endotoxin detection and their suitability for nanoparticulate materials. Conversely, the capacity of nanoparticles to bind endotoxin can be enhanced by functionalization with endotoxin-capturing molecules, opening the way to the development of novel endotoxin detection assays.

In the olive production sector, which is increasingly expanding beyond the borders of the Mediterranean basin, harvesting is the most demanding phase, from both an economic and organisational point of view. Traditional olive orchards are still predominant, with centuries-old and large plants, and are characterised by the gradual ripening of drupes and irregular planting patterns. Even though the structural conversion of these olive orchards into more modern cultivations may be difficult owing to their historical, monumental, and landscaping importance, as well as the existing legal restrictions, supporting a "modernisation" process aimed at mechanising the main farming operations remains a priority. Technological innovation is, therefore, a primary objective for Mediterranean olive growing, as well as for the enhancement of its strengths. The present study aimed at assessing different olive harvesting sites, considering the technical, economic, and environmental aspects, to develop a better version of the "olive harvesting database". The applied methodology, also called the "modular approach", represents a useful tool to apply in unitary process assessment to obtain a comprehensive database of diverse agricultural operations. Eight olive harvesting systems were compared: six highly mechanised scenarios, one based on mechanical-aided harvesting, and the final one based on fully manual harvesting. The mechanised systems obtained a better performance in terms of working capacity, as only 3.5 h ha -1 were needed to harvest 12 t using a self-propelled trunk shaker. In addition, the economic results revealed that mechanical harvesting, diversely from manual or aided harvesting, is the only way to decrease production costs. From an environmental point of view, manual and mechanical-aided harvesting showed the best performance in terms of impact per hour. However, using the mass-based unit (1 kg of harvested olives), the results were the opposite and this could be very relevant for the ecoprofile of olive oil., 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 Elsevier B.V. All rights reserved.)

Background: Limited and wide-ranging data are available on the recurrent Clostridioides difficile infection (rCDI) incidence rate., Methods: We performed a cohort study with the aim to assess the incidence of and risk factors for rCDI. Adult patients with a first CDI, hospitalized in 15 Italian hospitals, were prospectively included and followed-up for 30 d after the end of antimicrobial treatment for their first CDI. A case-control study was performed to identify risk factors associated with 30-day onset rCDI., Results: Three hundred nine patients with a first CDI were included in the study; 32% of the CDI episodes (99/309) were severe/complicated; complete follow-up was available for 288 patients (19 died during the first CDI episode, and 2 were lost during follow-up). At the end of the study, the crude all-cause mortality rate was 10.7% (33 deaths/309 patients). Two hundred seventy-one patients completed the follow-up; rCDI occurred in 21% of patients (56/271) with an incidence rate of 72/10,000 patient-days. Logistic regression analysis identified exposure to cephalosporin as an independent risk factor associated with rCDI (RR: 1.7; 95% CI: 1.1-2.7, p = 0.03)., Conclusion: Our study confirms the relevance of rCDI in terms of morbidity and mortality and provides a reliable estimation of its incidence.

Current legislation in Italy provides that medical Cannabis may be administered orally or by inhalation. One of the fundamental criteria for the administration of oral formulations is that they deliver a known consistent quantity of the active ingredients to ensure uniform therapies leading to the optimisation of the risks/benefits. In 2018, our group developed an improved Cannabis oil extraction technique. The objective of the present work was to carry out a stability study for the oil extracts obtained by this method. Furthermore, in order to facilitate the consumption of the prescribed medical Cannabis therapy by patients, a standard procedure was defined for the preparation of a single-dose preparation for oral use (hard capsules) containing the oil extract; thereafter, the quality and stability were evaluated. The hard capsules loaded with the oil extract were analysed and found to be uniform in content. The encapsulation process did not alter the quantity of the active molecule present in the oil. The stability tests yielded excellent results. Since the capsule dosage form is easily transported and administered, has pleasant organoleptic properties and is stable at room temperature for extended periods of time, this would facilitate the adherence to therapy by patients in treatment.

Understanding the modes of interaction between human monocytes/macrophages and engineered nanoparticles is the basis for assessing particle safety, in terms of activation of innate/inflammatory reactions, and their possible exploitation for medical applications. In vitro assessment of nanoparticle-macrophage interaction allows for examining the response of primary human cells, but the conventional 2D cultures do not reproduce the three-dimensional spacing of a tissue and the interaction of macrophages with the extracellular tissue matrix, conditions that shape macrophage recognition capacity and reactivity. Here, we have compared traditional 2D cultures with cultures on a 3D collagen matrix for evaluating the capacity gold nanoparticles to induce monocyte activation and subsequent innate memory in human blood monocytes in comparison to bacterial LPS. Results show that monocytes react to stimuli almost in the same way in 2D and 3D cultures in terms of production of TNFα and IL-6, but that notable differences are found when IL-8 and IL-1Ra are examined, in particular in the recall/memory response of primed cells to a second stimulation, with the 3D cultures showing cell activation and memory effects of nanoparticles better. In addition, the response variations in monocytes/macrophages from different donors point towards a personalized assessment of the nanoparticle effects on macrophage activation.

Herein, we demonstrate a cavity-enhanced hyperspectral refractometric imaging using an all-dielectric photonic crystal slab (PhCS). Our approach takes advantage of the synergy between two mechanisms, surface-enhanced fluorescence (SEF) and refractometric sensing, both based on high- Q resonances in proximity of bound states in the continuum (BICs). The enhanced local optical field of the first resonance amplifies of 2 orders of magnitude the SEF emission of a probe dye. Simultaneously, hyperspectral refractometric sensing, based on Fano interference between second mode and fluorescence emission, is used for mapping the spatially variant refractive index produced by the specimen on the PhCS. The spectral matching between first resonance and input laser is modulated by the specimen local refractive index, and thanks to the calibrated dependence with the spectral shift of the Fano resonance, the cavity tuning is used to achieve an enhanced correlative refractometric map with a resolution of 10 -5 RIU within femtoliter-scale sampling volumes. This is experimentally applied also on live prostate cancer cells grown on the PhCS, reconstructing enhanced surface refractive index images at the single-cell level. This dual mechanism of quasi-BIC spatially variant gain tracked by quasi-BIC refractometric sensing provides a correlative imaging platform that can find application in many fields for monitoring physical and biochemical processes, such as molecular interactions, chemical reactions, or surface cell analysis.

In the last decade, Raman Spectroscopy (RS) was demonstrated to be a label-free, non-invasive and non-destructive optical spectroscopy allowing the improvement in diagnostic accuracy in cancer and analytical assessment for cell sensing. This review discusses how Raman spectra can lead to a deeper molecular understanding of the biochemical changes in cancer cells in comparison to non-cancer cells, analyzing two key examples, leukemia and breast cancer. The reported Raman results provide information on cancer progression and allow the identification, classification, and follow-up after chemotherapy treatments of the cancer cells from the liquid biopsy. The key obstacles for RS applications in cancer cell diagnosis, including quality, objectivity, number of cells and velocity of the analysis, are considered. The use of multivariant analysis, such as principal component analysis (PCA) and linear discriminate analysis (LDA), for an automatic and objective assessment without any specialized knowledge of spectroscopy is presented. Raman imaging for cancer cell mapping is shown and its advantages for routine clinical pathology practice and live cell imaging, compared to single-point spectral analysis, are debated. Additionally, the combination of RS with microfluidic devices and high-throughput screening for improving the velocity and the number of cells analyzed are also discussed. Finally, the combination of the Raman microscopy (RM) with other imaging modalities, for complete visualization and characterization of the cells, is described.

Innate immune memory is characterized by a modulation in the magnitude with which innate immune cells such as monocytes and macrophages respond to potential dangers, subsequent to previous exposure to the same or unrelated agents. In this study, we have examined the capacity of gold nanoparticles (AuNP), which are already in use for therapeutic and diagnostic purposes, to modulate the innate memory induced by bacterial agents. The induction of innate memory was achieved in vitro by exposing human primary monocytes to bacterial agents (lipopolysaccharide -LPS-, or live Bacille Calmette-Guérin -BCG) in the absence or presence of AuNP. After the primary activation, cells were allowed to return to a resting condition, and eventually re-challenged with LPS. The induction of memory was assessed by comparing the response to the LPS challenge of unprimed cells with that of cells primed with bacterial agents and AuNP. The response to LPS was measured as the production of inflammatory (TNFα, IL-6) and anti-inflammatory cytokines (IL-10, IL-1Ra). While ineffective in directly inducing innate memory per se, and unable to influence LPS-induced tolerance memory, AuNP significantly affected the memory response of BCG-primed cells, by inhibiting the secondary response in terms of both inflammatory and anti-inflammatory factor production. The reprogramming of BCG-induced memory towards a tolerance type of reactivity may open promising perspectives for the use of AuNP in immunomodulatory approaches to autoimmune and chronic inflammatory diseases., Competing Interests: The authors declare no conflict of interest.

Under current legislation in Italy, Cannabis for medical purposes may be administered orally in the form of decoction or Cannabis oil extract. The scientific literature reports a number of preparation methods, mainly for oils, but no study is available that compares thoroughly, from a technological viewpoint, the Cannabis -based formulations currently administered to patients. With this in mind, this research work aimed to carry out specific formulation studies to design standard operating procedures for the preparation and optimization of Cannabis -based galenic formulations. Both decoctions and oils were prepared under different operating conditions to identify the most efficient process for the production of formulations with a high concentration of decarboxylated delta-9-tetrahydrocannabinol (THC) and cannabidiol (CBD). Regarding Cannabis oil, a new procedure has been developed that allows significantly higher recovery rates for THC and CBD compared with those for water-based extraction methods (decoction) and those for oil-based methods currently in use. Moreover, based on the results, it is possible to affirm that the prescription of Cannabis -based decoctions should not be the recommended first-choice solution for therapy, considering the low concentration of THC and CBD and, consequently, the high volume of decoction that the patient would have to ingest.

Advances in the adoption of technological innovations represent a great driver to improve the competitiveness of the Italian extra virgin olive oil (EVOO) industry. This work assesses the efficiency of an innovative extraction plant (with low oxidative impact, heating of paste before malaxation and a special decanter that avoids the final vertical centrifugation) in terms of oil yield and quality, and economic and environmental impacts. Economic and environmental impacts were evaluated by using both life cycle costing and life cycle assessment methodologies. A sensitivity analysis was also performed to highlight the uncertain factors that may strongly affect the results. Findings showed that olive milling with the innovative plant resulted in olive oil with a significant increase in quality, although the extraction yield was significantly higher when using conventional technology. In terms of environmental results, an average growth of 4.5% of the impacts in all categories was reached. The economic results revealed the highest extraction cost for the innovative scenario as well as the lower profitability, although a positive return in investment feasibility can be achieved due to an increase in the olive oil selling price. These findings could be useful to highlight the main hotspots in EVOO production and to suggest improvements for more sustainable management., Competing Interests: The authors declare no conflict of interest.

Rice ( Oryza sativa ) stands among the world's most important crop species. Rice is salt sensitive, and the undue accumulation of sodium ions (Na + ) in shoots has the strongest negative correlation with rice productivity under long-term salinity. The plasma membrane Na + /H + exchanger protein Salt Overly Sensitive 1 (SOS1) is the sole Na + efflux transporter that has been genetically characterized to date. Here, the importance of SOS1-facilitated Na + flux in the salt tolerance of rice was analyzed in a reverse-genetics approach. A sos1 loss-of-function mutant displayed exceptional salt sensitivity that was correlated with excessive Na + intake and impaired Na + loading into the xylem, thus indicating that SOS1 controls net root Na + uptake and long-distance Na + transport to shoots. The acute Na + sensitivity of sos1 plants at low NaCl concentrations allowed analysis of the transcriptional response to sodicity stress without effects of the osmotic stress intrinsic to high-salinity treatments. In contrast with that in the wild type, sos1 mutant roots displayed preferential down-regulation of stress-related genes in response to salt treatment, despite the greater intensity of stress experienced by the mutant. These results suggest there is impaired stress detection or an inability to mount a comprehensive response to salinity in sos1 In summary, the plasma membrane Na + /H + exchanger SOS1 plays a major role in the salt tolerance of rice by controlling Na + homeostasis and possibly contributing to the sensing of sodicity stress., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Introduction: Acute lumbosacral radicular syndrome is often a medical disorder of difficult management. Epidural steroid injection is a useful approach for the herniated disc and radiculitis. The transforaminal approach is usually considered more effective and target-specific, but it can be associated with permanent lower extremity paralysis. A caudal approach with an adjustable catheter has been widely used in adhesiolysis in chronic low back pain, but there are no reports of its application in acute radicular pain. The aim of this study is to assess the clinical effectiveness of epidural steroid injection by caudal approach with an adjustable catheter in patients with severe acute radicular pain., Methods: Fifty-five patients with severe acute radiculopathy were treated with epidural steroid injection by an epidural catheter whose tip can be directed laterally on the selected site. Numerical rating scale (NRS), pain relief, and analgesic consumption were observed after 1, 3, 6, and 12 months. Analgesic consumption (AC) and functional recovery (FR) have been considered secondary outcomes., Results: We observed a significant reduction of NRS score that was constant every 12 months. Pain relief was good after 1 month and improved further after 3 months. Only a few patients perceived poor pain relief and only three patients relapsed. More than 70% of the patients were drug-free at the 12th month., Conclusions: The caudal approach with adjustable catheter showed similar but more lasting effects on the acute severe radicular pain when compared to other epidural injections techniques; it is extremely target-specific and thus allows the use of small doses of corticosteroids; moreover, the adjustable catheter makes the procedure free from the risk of major complications., Funding: No funding or sponsorship was received for this study. Sponsorship for article publication fees were funded by TSS Medical SRL.

The paper describes the results of a specific LCA based analysis of the production of olive oil in the region of Calabria, in southern Italy. The goal of the study is to assess the energy and environmental impacts of different scenarios involving conventional and organic cultivations, plains and hills cultivations and involving different operating techniques. The study also aims at assessing the share of each life cycle step on the total of energy and environmental impacts. The functional unit chosen for the comparative analysis is a glass bottle of 0.75 L of extra virgin olive oil. A "from cradle to gate" perspective was chosen. The analysis was developed according to the LCA standards of the ISO 14040 series. The analysis is based on a field analysis developed in the last years in the province of Reggio Calabria between more than 50 enterprises and stakeholders of the field, representative of the whole Calabria region and of most southern Italy. The data used for the development of mass and energy balances are related to the years 2013-2015. The results clarify that for all indicators that the first part of the life cycle - from the production, including the growth of the olive plant to the full production stage - is the most relevant, variable between 80.6% share in the case of the particulate matter indicator to the 99.64% in the case of land use (Hill - Biological agriculture scenario). Relevant differences can be also traced for each specific indicator among all scenarios; high impacts are traced for the agricultural stages among all scenarios (70% -90% in all indicators) with high impacts caused by fertilizers. Among the transformation stages the bottle production is one of the most relevant sources of life cycle energy uses and environmental impacts (80-90%)., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Raman microspectroscopy (RM) and polarization sensitive digital holographic imaging (PSDHI) are valuable analytical tools in biological and medical research, allowing the detection of both biochemical and morphological variations of the sample without labels or long sample preparation. Here, using this multi-modal approach we analyze in vitro human sperm capacitation and the acrosome reaction induced by heparin. The multimodal microscopy provides morphofunctional information that can assess the sperms ability to respond to capacitation stimuli (sperm function). More precisely, the birefringence analysis in sperm cells can be used as an indicator of its structural normality. Indeed, digital holography applied for polarization imaging allows for revelation of the polarization state of the sample, showing a total birefringence of the sperm head in non-reacted spermatozoa, and a birefringence localized in the post-acrosomal region in reacted spermatozoa. Additionally, RM allows the detection and spectroscopic characterization of protein/lipid delocalization in the plasma and acrosomal membranes that can be used as valuable Raman biomarkers of sperm function. Interestingly, these spectral variations can be correlated with different time phases of the cell capacitation response. Although further experimentation is required, the proposed multimodal approach could represent a potential label-free diagnostic tool for use in reproductive medicine and the diagnosis of infertility.

Diatoms can represent the major component of phytoplankton and contribute massively to global primary production in the oceans. Over tens of millions of years they developed an intricate porous silica shell, the frustule, which ensures mechanical protection, sorting of nutrients from harmful agents, and optimization of light harvesting. Several groups of microalgae evolved different strategies of protection towards ultraviolet radiation (UVR), which is harmful for all living organisms mainly through the formation of dimeric photoproducts between adjacent pyrimidines in DNA. Even in presence of low concentrations of UV-absorbing compounds, several diatoms exhibit significant UVR tolerance. We here investigated the mechanisms involved in UVR screening by diatom silica investments focusing on single frustules of a planktonic centric diatom, Coscinodiscus wailesii, analyzing absorption by the silica matrix, diffraction by frustule ultrastructure and also UV conversion into photosynthetically active radiation exerted by nanostructured silica photoluminescence. We identified the defects and organic residuals incorporated in frustule silica matrix which mainly contribute to absorption; simulated and measured the spatial distribution of UVR transmitted by a single valve, finding that it is confined far away from the diatom valve itself; furthermore, we showed how UV-to-blue radiation conversion (which is particularly significant for photosynthetic productivity) is more efficient than other emission transitions in the visible spectral range.

Spores of the genus Bacillus are able to resist ionizing radiations and therefore they are a suitable biological model for studies in Astrobiology, i.e. the multidisciplinary approach to the study of the origin and evolution of life on Earth and in the universe. The resistance to γ-radiation is an important issue in Astrobiology in relation to the search for bacterial species that could adapt to life in space. This study investigates the resistance of spores of the thermophilic bacteria Parageobacillus thermantarcticus to γ-rays. The analysis of spores' response to irradiation at a molecular level is performed by means of Raman spectroscopy that allows to get insights in the sequence of events taking place during inactivation. The role of the γ-rays' dose in the inactivation of spores is also investigated, allowing to highlight the mechanism(s) of inactivation including DNA damage, protein denaturation and calcium dipicolinate levels.

Diatomite is a fossil material made of amorphous porous silica. In this work, polyethylene glycol (PEG)-modified diatomite NPs (PEG-DNPs) are decorated with gold NPs (AuNPs) by one-pot liquid-phase synthesis. Nanocomplexes (PEG-DNPs@AuNPs), with an average size of about 450 nm, are characterized by dynamic light scattering, electron microscopy, nitrogen adsorption/desorption analysis, UV-vis and photoluminescence spectroscopies. Preliminary studies on the use of the nanocomplex in nanomedicine are also presented. Tests performed incubating PEG-DNPs@AuNPs in physiological conditions reveal a good stability of material. Cellular uptake of labeled PEG-DNPs@AuNPs is investigated by confocal microscopy after incubation with human cervix epithelioid carcinoma (HeLa) cells up to 48 h: an efficient cytoplasmic localization is observed. In vitro cytotoxicity of nanocomplexes with a concentration up to 400 μg ml -1 for 72 h is also evaluated. The results suggest the use of PEG-DNPs@AuNPs as advanced nanodevices adding imaging features to the nanocomplexes, due to AuNPs as contrast agent.

The mechanization of farming operation plays an important role in improving the profitability of the agricultural sector by increasing work productivity and reducing production costs. However, the new challenges of agriculture also include the environmental issues. The choice between different alternatives to perform a determined agricultural practice should be based on reliable information, considering technical, economic and environmental aspects. Olive growing represents the most important agricultural production in the Mediterranean Basin and its mechanization, particularly harvesting, could have major impacts on the sustainability of this production. This study aims at assessing various olive-harvesting scenarios, while considering technical, economic and environmental aspects in order to build a beta version of the "olive-harvesting database". The proposed methodology called "modular approach" could represent a useful tool to apply in unitary process assessment in order to obtain a comprehensive database of the diverse agricultural operations. The methodology was based on Life Cycle Assessment and production cost analysis. Technical performance evaluation showed that the recorded work capacities varied between 5 tons of harvested olives per day when employing mechanical harvest aids and 18 tons per day when employing trunk shakers. The economic evaluation highlighted that the harvesting costs are variable as a function of the given cost type (costs per hour, costs per kg of harvested olives and costs per hectare). The LCA revealed that mechanically aided techniques were the most sustainable ones when the functional unit is considered as one harvesting hour, although this FU is not the most suitable unit for choosing the best environmental solution. The surface and production mass units are more appropriate FUs in comparative studies, although they are strictly linked to the "work capacity". A significant variation in the environmental performances depended on the FUs and on the average yields when the FU represented one kg of harvested olives., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Cation antiporters of the NHX family are widely regarded as determinants of salt tolerance due to their capacity to drive sodium (Na) and sequester it into vacuoles. Recent work shows, however, that NHX transporters are primarily involved in vacuolar potassium (K) storage. Over-expression of the K/H antiporter AtNHX1 in tomato increases K accumulation into vacuoles and plant sensitivity to K deprivation. Here we show that the appearance of early leaf symptoms of K deficiency was associated with higher concentration of polyamines. Transgenic roots exhibited a greater sensitivity than shoots to K deprivation with changes in the composition of the free amino acids pool, total sugars and organic acids. Concentrations of amides (glutamine), amino acids (arginine) and sugars significantly increased in root, together with a reduction in malate and succinate concentrations. The concentration of pyruvate and the activity of pyruvate kinase were greater in the transgenic roots before K withdrawal although both parameters were depressed by K deprivation and approached wild-type levels. In the longer term, the over-expression of the NHX1 antiporter affected root growth and biomass partitioning (shoot/root ratio). Greater ethylene release produced longer stem internodes and leaf curling in the transgenic line. Our data show that enhanced sequestration of K by the NHX antiporter in the vacuoles altered cellular K homeostasis and had deeper physiological consequences than expected. Early metabolic changes lead later on to profound morphological and physiological adjustments resulting eventually in the loss of nutrient use efficiency., (© 2017 Scandinavian Plant Physiology Society.)

The analysis of leukocytes of peripheral blood is a crucial step in hematologic exams commonly used for disease diagnosis and, typically, requires molecular labelling. In addition, only a detailed, laborious phenotypic analysis allows identifying the presence and stage of specific pathologies such as leukemia. Most of the biochemical information is lost in the routine blood tests. In the present study, we tackle 2 important issues of label-free biochemical identification and classification of leukocytes using Raman spectroscopy (RS). First, we demonstrate that leukocyte subpopulations of lymphocytes (B, T and NK cells), monocytes and granulocytes can be identified by the unsupervised statistical approach of principal component analysis and classified by linear discriminant analysis with approximately 99% of accuracy. Second, we apply the same procedure to identify and discriminate normal B cells and transformed MN60 lymphocyte leukemic cell lines. In addition, we demonstrate that RS can be efficiently used for monitoring the cell response to low-dose chemotherapy treatment, experimentally eliciting the sensitivity to a dose-dependent cell response, which is of fundamental importance to determine the efficacy of any treatment. These results largely expand established Raman-based research protocols for label-free analysis of white blood cells, leukemic cells and chemotherapy treatment follow-up., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this work, we propose the use of complex, bioderived nanostructures as efficient surface-enhanced Raman scattering (SERS) substrates for chemical analysis of cellular membranes. These structures were directly obtained from a suitable gold metalization of the Pseudonitzchia multistriata diatom silica shell (the so called frustule), whose grating-like geometry provides large light coupling with external radiation, whereas its extruded, subwavelength lateral edge provides an excellent interaction with cells without steric hindrance. We carried out numerical simulations and experimental characterizations of the supported plasmonic resonances and optical near-field amplification. We thoroughly evaluated the SERS substrate enhancement factor as a function of the metalization parameters and finally applied the nanostrucures for discriminating cell membrane Raman signals. In particular, we considered two cases where the membrane composition plays a fundamental role in the assessment of several pathologies, that is, red blood cells and B-leukemia REH cells.

Porous biosilica nanoparticles obtained from diatomites (DNPs) have been recently demonstrated to be non-toxic nanovectors of therapeutic agents in cancer cells. In this work, the internalization kinetics and intracellular spatial distribution of functionalized DNPs incubated with human lung epidermoid carcinoma cell line (H1355) up to 72 hours are investigated by Raman imaging. The label-free Raman results are compared with confocal fluorescence microscopy and photoluminescence (PL) data. Raman bands specifically assigned to DNPs and cellular components provide evidence that the nanovectors are internalized and co-localize with lipid environments. A considerable DNPs uptake in cells is observed within 6 hours, with equilibrium being achieved after 18 hours. The obtained data show the presence of DNPs up to 72 hours, without damage to cell viability or morphology. The PL measurements performed on DNPs not penetrating the cells at different incubation times are strongly correlated with the results obtained by Raman imaging and confocal microscopy analyses., (© 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In this paper we report on the engineering of repeatable surface enhanced Raman scattering (SERS) optical fiber sensor devices (optrodes), as realized through nanosphere lithography. The Lab-on-Fiber SERS optrode consists of polystyrene nanospheres in a close-packed arrays configuration covered by a thin film of gold on the optical fiber tip. The SERS surfaces were fabricated by using a nanosphere lithography approach that is already demonstrated as able to produce highly repeatable patterns on the fiber tip. In order to engineer and optimize the SERS probes, we first evaluated and compared the SERS performances in terms of Enhancement Factor (EF) pertaining to different patterns with different nanosphere diameters and gold thicknesses. To this aim, the EF of SERS surfaces with a pitch of 500, 750 and 1000 nm, and gold films of 20, 30 and 40 nm have been retrieved, adopting the SERS signal of a monolayer of biphenyl-4-thiol (BPT) as a reliable benchmark. The analysis allowed us to identify of the most promising SERS platform: for the samples with nanospheres diameter of 500 nm and gold thickness of 30 nm, we measured values of EF of 4 × 10⁵, which is comparable with state-of-the-art SERS EF achievable with highly performing colloidal gold nanoparticles. The reproducibility of the SERS enhancement was thoroughly evaluated. In particular, the SERS intensity revealed intra-sample (i.e., between different spatial regions of a selected substrate) and inter-sample (i.e., between regions of different substrates) repeatability, with a relative standard deviation lower than 9 and 15%, respectively. Finally, in order to determine the most suitable optical fiber probe, in terms of excitation/collection efficiency and Raman background, we selected several commercially available optical fibers and tested them with a BPT solution used as benchmark. A fiber probe with a pure silica core of 200 µm diameter and high numerical aperture (i.e., 0.5) was found to be the most promising fiber platform, providing the best trade-off between high excitation/collection efficiency and low background. This work, thus, poses the basis for realizing reproducible and engineered Lab-on-Fiber SERS optrodes for in-situ trace detection directed toward highly advanced in vivo sensing., Competing Interests: The authors declare no conflict of interest.

Traditionally, palliative care (PC) systems focused on the needs of advanced cancer patients, but most patients needing PC have end-stage organ diseases. Similarly, PC models focus on the needs of patients in hospices or at home; however, in most cases PC is provided in acute hospitals. Indeed, the symptom burden that these patients experience in the last year of life frequently forces them to seek care in emergency departments. The majority of them are admitted to the hospital and many die. This issue poses important concerns. Despite the efforts of attending healthcare professionals, in-hospital patients do not receive optimal care near the end-of-life. Also, evidence is emerging that delay in identifying patients needing PC have a detrimental impact on their quality of life (QoL). Therefore, there is an urgent need to identify, early and properly, these patients among those hospitalized. Several trials reported the efficacy of PC in improving the QoL in these patients. Each hospital should ensure that a multidisciplinary PC team is available to support attending physicians to achieve the best QoL for both PC patients and their families. This review discusses the role and the impact of in-hospital PC in patients with end-stage disease or advanced cancer., Competing Interests: The authors declare no conflict of interest.

Life cycle (LC) methodologies have attracted a great interest in agricultural sustainability assessments, even if, at the same time, they have sometimes been criticized for making unrealistic assumptions and subjective choices. To cope with these weaknesses, Multi-Criteria Decision Analysis (MCDA) and/or participatory methods can be used to balance and integrate different sustainability dimensions. The purpose of this study is to highlight how life cycle approaches were combined with MCDA and participatory methods to address agricultural sustainability in the published scientific literature. A systematic and critical review was developed, highlighting the following features: which multi-criterial and/or participatory methods have been associated with LC tools; how they have been integrated or complemented (methodological relationships); the intensity of the involvement of stakeholders (degree of participation); and which synergies have been achieved by combining the methods. The main typology of integration was represented by multi-criterial frameworks integrating LC evaluations. LC tools can provide MCDA studies with local and global information on how to reduce negative impacts and avoid burden shifts, while MCDA methods can help LC practitioners deal with subjective assumptions in an objective way, to take into consideration actors' values and to overcome trade-offs among the different dimensions of sustainability. Considerations concerning the further development of Life Cycle Sustainability Assessment (LCSA) have been identified as well., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Background: The aims of this study were to evaluate the feasibility of an Emergency Department (ED)-initiated screening to identify seriously ill patients in need of palliative care (PC) and to develop a simplified screening tool (SST)., Methods: Eligible patients with known diagnosis of chronic heart, lung, liver, and kidney failures, progressive neurological diseases or advanced cancer, awaiting to be hospitalized after an ED visit, were assessed with the screening tool from the Italian Society of Anesthesia, Analgesia, Resuscitation, and Intensive Care (SIAARTI)., Results: Out of 1497 patients with an ED visit, 485 were hospitalized, and 257 of them met the inclusion criteria. Of 257 enrolled patients, 91 (35%) were identified as in need of PC. Comparing patients with 4 positive criteria to those with <4, the general clinical indicators more frequently positive were: ≥1 admission within the last 12 months (P<0.001); hospital admission from or awaiting admission to health care services (HCS)/Hospice (P<0.001); cachexia (P<0.012); home oxygen use (P<0.001); dialysis (P<0.008). A SST was developed to identify patients in need of PC when a Palliative Performance Scale score <50 was present with at least one of the following indicators: ≥1 admission within the last 12 months; hospital admission from HCS; awaiting admission to HCS/Hospice; dialysis; home oxygen use; non-invasive ventilation. This SST showed a good agreement with the SIAARTI one as sensitivity (97.8%), specificity (92.8%), and accuracy (94.5%)., Conclusions: Our study estimated that over one-third of the people with chronic diseases awaiting to be hospitalized after an ED visit were in need of PC and can be identified with this easy-to-use, non-disease-specific SST.

Acute lymphoblastic leukemia type B (B-ALL) is a neoplastic disorder that shows high mortality rates due to immature lymphocyte B-cell proliferation. B-ALL diagnosis requires identification and classification of the leukemia cells. Here, we demonstrate the use of Raman spectroscopy to discriminate normal lymphocytic B-cells from three different B-leukemia transformed cell lines (i.e., RS4;11, REH, MN60 cells) based on their biochemical features. In combination with immunofluorescence and Western blotting, we show that these Raman markers reflect the relative changes in the potential biological markers from cell surface antigens, cytoplasmic proteins, and DNA content and correlate with the lymphoblastic B-cell maturation/differentiation stages. Our study demonstrates the potential of this technique for classification of B-leukemia cells into the different differentiation/maturation stages, as well as for the identification of key biochemical changes under chemotherapeutic treatments. Finally, preliminary results from clinical samples indicate high consistency of, and potential applications for, this Raman spectroscopy approach.

Purpose: Pain is a common symptom in cancer patients and often the most tangible sign of disease they and their families perceive. Despite currently available treatments, cancer pain frequently remains underrated and undertreated because of lack of adherence to the prescribed drug regimen. With this study we sought to identify elements that could facilitate pain management by exploring through narrative interviews the lived experiences of patients with severe chronic cancer pain in relation to their adherence to pain therapy., Method: A purposive sample of 18 cancer patients, treated at the Centre for Oncology and Haematology (COES), City Hospital for Health and Science, Turin, were interviewed. The interview contents were analysed using a qualitative phenomenological methodology as described by Giorgi., Results: Three themes emerged from analysis of the interview transcripts: the significance of pain in subjective experience; the experience of being a patient pursuing a care pathway and the importance attributed to pain therapy. Factors facilitating adherence included the perception of the physical and psychological benefits of having and following a pain medications plan, subjective self-efficacy in pain control, and trust in the healthcare team. Barriers to adherence were negative attitudes toward opioid analgesic therapy, debilitating drug side effects, and denial of pain as a tangible sign of disease., Conclusion: Probing into the significance of the pain experience and its treatment through these narrative interviews revealed several core constituents of adherence. Healthcare providers can use this better understanding to build a trusting relationship with patients and foster adherence to treatment throughout the care pathway., (Copyright © 2015 Elsevier Ltd. All rights reserved.)