Inverse regulation of SOS1 and HKT1 protein localization and stability by SOS3/CBL4 in Arabidopsis thaliana.

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.

High throughput clogging free microfluidic particle filter by femtosecond laser micromachining.

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).

Combined SERS-Raman screening of HER2-overexpressing or silenced breast cancer cell lines.

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.

Application of Raman spectroscopy to the evaluation of F-actin changes in sea urchin eggs at fertilization.

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 Ca2+ 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 Ca2+ 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.

SERS Quantification of Galunisertib Delivery in Colorectal Cancer Cells by Plasmonic-Assisted Diatomite Nanoparticles.

The small molecule Galunisertib (LY2157299, LY) shows multiple anticancer activities blocking the transforming growth factor-ß1 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.

A Critical Role of Sodium Flux via the Plasma Membrane Na+/H+ Exchanger SOS1 in the Salt Tolerance of Rice.

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.

Raman Microscopy: Progress in Research on Cancer Cell Sensing.

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.

Life cycle assessment of olive oil: A case study in southern Italy.

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%).

Resistance and Raman spectroscopy analysis of Parageobacillus thermantarcticus spores after γ-ray exposure.

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.

Pleiotropic effects of enhancing vacuolar K/H exchange in tomato.

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.

Gold decorated porous biosilica nanodevices for advanced medicine.

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.

Nanosphere Lithography on Fiber: Towards Engineered Lab-On-Fiber SERS Optrodes.

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.

Ion exchangers NHX1 and NHX2 mediate active potassium uptake into vacuoles to regulate cell turgor and stomatal function in Arabidopsis.

Intracellular NHX proteins are Na(+),K(+)/H(+) antiporters involved in K(+) homeostasis, endosomal pH regulation, and salt tolerance. Proteins NHX1 and NHX2 are the two major tonoplast-localized NHX isoforms. Here, we show that NHX1 and NHX2 have similar expression patterns and identical biochemical activity, and together they account for a significant amount of the Na(+),K(+)/H(+) antiport activity in tonoplast vesicles. Reverse genetics showed functional redundancy of NHX1 and NHX2 genes. Growth of the double mutant nhx1 nhx2 was severely impaired, and plants were extremely sensitive to external K(+). By contrast, nhx1 nhx2 mutants showed similar sensitivity to salinity stress and even greater rates of Na(+) sequestration than the wild type. Double mutants had reduced ability to create the vacuolar K(+) pool, which in turn provoked greater K(+) retention in the cytosol, impaired osmoregulation, and compromised turgor generation for cell expansion. Genes NHX1 and NHX2 were highly expressed in guard cells, and stomatal function was defective in mutant plants, further compromising their ability to regulate water relations. Together, these results show that tonoplast-localized NHX proteins are essential for active K(+) uptake at the tonoplast, for turgor regulation, and for stomatal function.

Modulated Raman Spectroscopy for Enhanced Cancer Diagnosis at the Cellular Level.

Raman spectroscopy is emerging as a promising and novel biophotonics tool for non-invasive, real-time diagnosis of tissue and cell abnormalities. However, the presence of a strong fluorescence background is a key issue that can detract from the use of Raman spectroscopy in routine clinical care. The review summarizes the state-of-the-art methods to remove the fluorescence background and explores recent achievements to address this issue obtained with modulated Raman spectroscopy. This innovative approach can be used to extract the Raman spectral component from the fluorescence background and improve the quality of the Raman signal. We describe the potential of modulated Raman spectroscopy as a rapid, inexpensive and accurate clinical tool to detect the presence of bladder cancer cells. Finally, in a broader context, we show how this approach can greatly enhance the sensitivity of integrated Raman spectroscopy and microfluidic systems, opening new prospects for portable higher throughput Raman cell sorting.

The effectiveness of the quality program Pac-IficO to improve pain management in hospitalized cancer patients: a before-after cluster phase II trial.

BACKGROUND: Cancer-related pain continues to be a major healthcare issue worldwide. Despite the availability of effective analgesic drugs, published guidelines and educational programs for Health Care Professionals (HCPs) the symptom is still under-diagnosed and its treatment is not appropriate in many patients. The objective of the study is to evaluate the efficacy of the Pac-IFicO programme in improving the quality of pain management in hospitalised cancer patients. METHODS/DESIGN: This is a before-after cluster phase II study. After the before assessment, the experimental intervention - the Pac-IFicO programme - will be implemented in ten medicine, oncology and respiratory disease hospital wards. The same assessment will be repeated after the completion of the intervention. The Pac-IFicO programme is a complex intervention with multiple components. It includes focus group with ward professionals for identifying possible local obstacles to optimal pain control, informative material for the patients, an educational program performed through guides from the wards, and an organisational intervention to the ward. The primary end-point of the study is the proportion of cancer patients with severe pain. Secondary end-points include opioids administered in the wards, knowledge in pain management, and quality of pain management. We plan to recruit about 500 cancer patients. This sample size should be sufficient, after appropriate statistical adjustments for clustering, to detect an absolute decrease in the primary end-point from 20% to 9%. DISCUSSION: This trial is aimed at exploring with an experimental approach the efficacy of a new quality improvement educational intervention. TRIAL REGISTRATION: ClinicalTrials.gov: NCT02035098.

Correlative Raman Imaging: Development and Cancer Applications.

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.

Towards a New Generation of Hormone Therapies: Design, Synthesis and Biological Evaluation of Novel 1,2,3-Triazoles as Estrogen-Positive Breast Cancer Therapeutics and Non-Steroidal Aromatase Inhibitors.

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, 13C NMR, 1H 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.

A lentiviral vector-based, herpes simplex virus 1 (HSV-1) glycoprotein B vaccine affords cross-protection against HSV-1 and HSV-2 genital infections.

Genital herpes is caused by herpes simplex virus 1 (HSV-1) and HSV-2, and its incidence is constantly increasing in the human population. Regardless of the clinical manifestation, HSV-1 and HSV-2 infections are highly transmissible to sexual partners and enhance susceptibility to other sexually transmitted infections. An effective vaccine is not yet available. Here, HSV-1 glycoprotein B (gB1) was delivered by a feline immunodeficiency virus (FIV) vector and tested against HSV-1 and HSV-2 vaginal challenges in C57BL/6 mice. The gB1 vaccine elicited cross-neutralizing antibodies and cell-mediated responses that protected 100 and 75% animals from HSV-1- and HSV-2-associated severe disease, respectively. Two of the eight fully protected vaccinees underwent subclinical HSV-2 infection, as demonstrated by deep immunosuppression and other analyses. Finally, vaccination prevented death in 83% of the animals challenged with a HSV-2 dose that killed 78 and 100% naive and mock-vaccinated controls, respectively. Since this FIV vector can accommodate two or more HSV immunogens, this vaccine has ample potential for improvement and may become a candidate for the development of a truly effective vaccine against genital herpes.

Cancer metabolic features allow discrimination of tumor from white blood cells by label-free multimodal optical imaging.

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.

Profilin 1 deficiency drives mitotic defects and reduces genome stability.

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.