Search

Introduction: Plasmapheresis donation is considered safe and well tolerated, although long-term effects need to be clarified. The volumes of anticoagulant (ACD-A) used are variable and depend primarily on hematocrit (HCT), total blood processed, amount of plasma collected, and donor characteristics. To elucidate the effect of the plasma unit weight setting on plasmapheresis efficiency and ACD-A distribution, we enrolled male donors undergoing a controlled apheresis process donating 700 g and 720 g in two different sessions. In parallel, we investigated a possible effect of sex, recruiting women donating 700 g of plasma. Methods: The study was conducted on men donating 720 g and (12 months later) 700 g of plasma, and on women donating 700 g of plasma. The main outcomes were pre-/post-donation delta (Δ) citrate concentration in donor plasma and ACD-A reinfused to the donor. Information concerning the annual check-up and the procedure was also collected. Intergroup comparisons (men donating 720 g vs. men donating 700 g and men vs. women both donating 700 g) and intragroup associations with donor and procedural characteristics were reported. Results: With the procedure set at 720 g, the machine processed around 44 mL more whole blood to collect 20 g more plasma, and 720 g donors received around 12 mL more anticoagulant than 700 g donors. Accordingly, Δ citrate concentration was 1.5 times higher (12 μm), with a greater variability observed for 720 g donations. Citrate concentration in the plasma unit was lower in the 720 g group, although not significantly. Comparing outcomes between women and men donating 700 g, we observed higher (and highly variable) Δ citrate and reinfused ACD-A in women, accompanied by lower anticoagulant levels in the unit. Increased Δ citrate is inversely associated with HCT and age in men and with HCT and triglycerides in women. Reinfused ACD-A correlates with HCT in women but not in men. Conclusion: Unit weight setting and sex influence an ACD-A shift from the estimated values toward an increased reinfusion to donor. In parallel, we observed an impact of age and sex on post-donation citrate metabolism. Altogether, these elements should be taken into account for the development of tailored approaches aimed at maintaining similar safety profiles for all donors using different plasmapheresis settings.

Serum magnesium is the most frequently used laboratory test for evaluating clinical magnesium status. Hypomagnesemia (low magnesium status), which is associated with many chronic diseases, is diagnosed using the serum magnesium reference range. Currently, no international consensus for a magnesemia normal range exists. Two independent groups designated 0.85 mmol/L (2.07 mg/dL; 1.7 mEq/L) as the low cut-off point defining hypomagnesemia. MaGNet discussions revealed differences in serum magnesium reference ranges used by members' hospitals and laboratories, presenting an urgent need for standardization.We gathered and compared serum magnesium reference range values from our institutions, hospitals, and colleagues worldwide.Serum magnesium levels designating "hypomagnesemia" differ widely. Of 43 collected values, only 2 met 0.85 mmol/L as the low cut-off point to define hypomagnesemia. The remainder had lower cut-off values, which may underestimate hypomagnesemia diagnosis in hospital, clinical, and research assessments. Current serum magnesium reference ranges stem from "normal" populations, which unknowingly include persons with chronic latent magnesium deficit (CLMD). Serum magnesium levels of patients with CLMD fall within widely used "normal" ranges, but their magnesium status is too low for long-term health. The lower serum magnesium reference (0.85 mmol/L) proposed specifically prevents the inclusion of patients with CLMD.Widely varying serum magnesium reference ranges render our use of this important medical tool imprecise, minimizing impacts of low magnesium status or hypomagnesemia as a marker of disease risk. To appropriately diagnose, increase awareness of, and manage magnesium status, it is critical to standardize lower reference values for serum magnesium at 0.85 mmol/L (2.07 mg/dL; 1.7 mEq/L).

Several ABO gene mutations are known to determine rare subgroups: these ABO variants are often responsible for weak or null phenotypes and may cause an incorrect determination of the serotype. Here we describe for the first time the phenotypic discrepancy of a rare B allele within the same Caucasian family that depends on the co-inheritance with A or H antigen. Blood samples from newborns, mothers, and grandmothers were analysed through routine serotype and genotype testing. Blood compatibility test was performed for red blood cells or serum of the grandmother. ABO exons were investigated through PCR and sanger sequencing. According to serology, the phenotype of the mother was AB, while it was O for the newborn. Genotype analysis confirmed that the mother was AB, while the newborn was found to be B. Sanger sequencing revealed the presence of a rare mutation in both individuals (784 GA, D262N), corresponding to the ABO*BW.17 allele. The grandmother was found to have the same genotype/serotype of the newborn. Crossmatch testing suggested that subjects with this genotype/serotype might be considered O donors and recipients.

Various ocular surface diseases are treated with blood-derived eye drops. Their use has been introduced in clinical practice because of their metabolite and growth factor content, which promotes eye surface regeneration. Blood-based eye drops can be prepared from different sources (i.e., whole blood or platelet apheresis donation), as well as with different protocols (e.g., different dilutions and freeze/thaw cycles). This variability hampers the standardization of clinical protocols and, consequently, the evaluation of their clinical efficacy. Detailing and sharing the methodological procedures may contribute to defining common guidelines. Over the last years, allogenic products have been diffusing as an alternative to the autologous treatments since they guarantee higher efficacy standards; among them, the platelet-rich plasma lysate (PRP-L) eye drops are prepared with simple manufacturing procedures. In the transfusion medicine unit at AUSL-IRCCS di Reggio Emilia, Italy, PRP-L is obtained from platelet-apheresis donation. This product is initially diluted to 0.3 x 10

Background X-ray Fluorescence (XRF) microscopy has become a widespread method to observe the location of elemental components within cell. Although it is becoming a widely used technique in many laboratories and synchrotron facilities, sample preparation for this microscopy remains one of the main concerns. Indeed, the information available to researchers on how to treat samples are insufficient and not clear, sometimes leading to waste of time and/or compromising the success of the experiment. It is indeed recognised that a correct sample preparation is the first important step in performing a successful and meaningful analysis, i.e. achieving a condition in which the cells are closer to their physiological state. The majority of the commonly used protocols of cell fixation for X-ray microscopy have been mainly imported and adapted from immunocytochemistry. Therefore, many fixation methods are described, but none of them have been tested in a systematic way and declared the most eligible for synchrotron-based X-ray microscopy. Methods HEC-1-A endometrial cell line, human spermatozoa and Human Embryonic Kidney HEK-293 cell line were chosen as cellular models; all type of cells were fixed using both organic solvents and cross-linking methods, as follow: 70% ethanol, paraphormaldehyde at 3.7 % and 2%; in addition HEK-293 cells were also subjected to methanol/acetone treatment and to cryofixation. Comparison among the different fixation methods was conducted by coupling Low Energy X-ray fluorescence (LEXRF), with Scanning Transmission X-ray Microscopy (STXM) and Atomic Force Microscopy (AFM). Results Organic solvents induce greater cellular dehydration, with the maximal impact on the distribution and depletion of the diffusive elements. Paraphormaldehyde seems to be able to provide robust and replicable data. Finally, the cryofixed cells give the best results both in terms of morphology and elemental content. Conclusion Although cryofixation appears to be the most appropriate method as it allows the cell to be kept closer to physiological conditions, it has some technical limitations. PFA, when used at conventional concentration of 3.7%, is also an excellent alternative method for X-ray microscopy.

Low-density lipoprotein (LDL) apheresis (LA) selectively eliminates lipoproteins containing apolipoprotein B 100 (ApoB100) on patients affected by severe dyslipidemia. In addition to lowering lipids, LA is thought to exert pleiotropic effects altering a number of other compounds associated with atherosclerosis, such as pro- and anti-inflammatory cytokines or pro-thrombotic factors. More knowledge needs to be gathered on the effects of LA, and particularly on its ability to modify blood components other than lipids. We performed a multiparametric assessment of the inflammatory, metabolic and proteomic profile changes after Heparin-induced lipoprotein precipitation (H.E.L.P.) apheresis on serum samples from nine dyslipidemic patients evaluating cholesterol and lipoproteins, plasma viscosity and density, metabolites, cytokines, PCSK9 levels and other proteins selectively removed after the treatment. Our results show that H.E.L.P. apheresis is effective in lowering lipoprotein and PCSK9 levels. Although not significantly, complement and inflammation-related proteins are also affected, indicating a possible transient epiphenomenon induced by the extracorporeal procedure.

Background and Objectives The first wave of coronavirus disease‐2019 (COVID‐19) dramatically affected the Transfusion Medicine Unit of the Azienda Unità Sanitari Locale ‐ Istituto di Ricovero e Cura a Carattere Scientifico (AUSL‐IRCCS) di Reggio Emilia, which faced a total rearrangement of the procedures for donors and patients. This study aims to assess the major implications of COVID‐19 on our department, focusing on the blood transfusion chain and therapies, in order to support transfusion specialists in seeking efficient ways to face similar future emergencies. Materials and Methods This retrospective study compares our Transfusion Medicine Unit data collected between February and May 2020 with the same period in 2017–2019. Data on red blood cells and platelets donations, transfusions and clinical procedures were collected as aggregates from our internal electronic database. Results During the lockdown, donor centres were re‐organized to reduce the risk of contagion and avoid unnecessary blood collection. Blood donations were re‐scheduled to meet the decrease in elective surgery; consequently, plateletapheresis was implemented to supply the reduction of buffycoat‐derived platelets. Transfusions significantly decreased together with orthopaedic and vascular surgery, while they were only marginally diminished for both cancer and onco‐haematological patients. Reduced procedures for inpatients and outpatients were matched by remote medicine, addressing the need of a constant healthcare support for patients with chronic diseases. Conclusions The described measures were adopted to avoid excessive blood collection and expiration, guarantee the safety of our ward (for both patients and staff) and supply the necessary transfusion therapies. These measures may support the development of appropriate risk management plans and safety procedures for other hospitals and transfusion services that have to face similar events.

The quantitative uptake of Silica nanoparticles (SiNPs), although representing an essential prerequisite for their theranostic use, is difficult to address and it is still not utterly investigated. In this study, we tested the uptake and toxicity of two different types of luminescent core-shell silica-PEG (polyethylene glycol) nanoparticles SiNP and their carboxylate analogues on human adenocarcinoma cell line LoVo. We assessed the intracellular spatial distribution and concentration of Si element in the cell by a state-of-the-art approach merging synchrotron-based X-ray techniques (XRFM) with scanning transmission X-Ray microscopy (STXM). The concentration maps of Si obtained reflect the distribution of the SiNPs. In addition, we calculated the number of SiNPs per volume unit in each single cell, quantitating the exact amount of conveyed particles. The absence of effects on proliferation and cell death was confirmed by viability assays, morphological analysis and cytofluorimetric evaluation of ROS content. The three-dimensional analysis of intracellular uptake of both types of nanoparticles (with different surface charge) was performed by confocal fluorescence microscopy, which showed a main localization in the cytosolic region with no sign of nuclear uptake.

Background: Increasing evidences support a correlation between magnesium (Mg) homeostasis and colorectal cancer (CRC). Nevertheless, the role of Mg and its transporters as diagnostic markers in CRC is still a matter of debate. In this study we combined X-ray Fluorescence Microscopy and databases information to investigate the possible correlation between Mg imbalance and CRC. Methods: CRC tissue samples and their non-tumoural counterpart from four patients were collected and analysed for total Mg level and distribution by X-Ray Fluorescence Microscopy. We also reviewed the scientific literature and the main tissue expression databases to collect data on Mg transporters expression in CRC. Results: We found a significantly higher content of total Mg in CRC samples when compared to non-tumoural tissues. Mg distribution was also impaired in CRC. Conversely, we evidenced an uncertain correlation between Mg transporters expression and colon malignancies. Discussion: Although further studies are necessary to determine the correlation between different cancer types and stages, this is the first report proposing the measurement of Mg tissue localisation as a marker in CRC. This study represents thus a proof-of-concept that paves the way for the design of a larger prospective investigation of Mg in CRC.

When the current SARS-CoV-2 pandemic began in early 2020, the global magnesium researcher community came together and noted the striking similarities between COVID-19 risk factors and conditions associated with magnesium deficit state in humans, reasoning that magnesium deficiency could worsen the course of COVID-19 [1-4]. This prompted establishment of a worldwide collaborative network with regular virtual meetings to brainstorm the associations between magnesium and COVID-19. We hypothesize that magnesium deficiency, a common but mostly unrecognized state in modern global societies, could be an important component of the susceptibility to SARS-CoV-2 infection. Consequently, restoring the magnesium deficit may be a putative therapeutic strategy to possibly ameliorate or prevent COVID-19.

BACKGROUND: Red blood cell (RBC) units may contain a variety of molecules that can activate the neutrophil cascade turning neutrophils into targets for immunomodulatory molecules. Our metabolomics profiling of RBC units revealed a significant increase of hypoxanthine concentration during storage. Hypoxanthine catabolism in vivo ends with the production of uric acid through a reaction catalysed by xanthine oxidase during which reactive oxygen species are generated. Some authors have described in vitro neutrophil activation after treatment with stored RBC medium. However, the response of neutrophils to the action of xanthine oxidase upon hypoxanthine accumulation in the supernatant of RBC units has never been investigated. MATERIALS AND METHODS: Neutrophils were isolated from peripheral whole blood and cultured at 37 °C in a humidified incubator with 5% CO(2). Hypoxanthine and RBC supernatants were tested to verify neutrophil stimulation. To prove the involvement of hypoxanthine in neutrophil activation, xanthine oxidase was pre-incubated with or without allopurinol before addition to the neutrophil cultures. Intracellular expression of tumour necrosis factor-α (TNF-α) and interleukin-8 (IL-8) was assessed by a cytofluorimetric assay and early-stage release of IL-8 was detected by a Luminex(®) assay. RESULTS: In the presence of xanthine oxidase, hypoxanthine, alone and in combination with RBC supernatants, caused increases of TNF-α- and IL-8-positive cells after 5 hours of treatment. Moreover, IL-8 was quickly released, 30 min after stimulation. DISCUSSION: Here we show, for the first time, that neutrophil activation by stored RBC depends, in part, on the presence of hypoxanthine contained in the RBC units. Our results add hypoxanthine to the already known mediators of inflammation present in RBC units, supporting the evidence that medium from stored RBC may concur to boost inflammatory processes in transfusion recipients, potentially leading to negative post-transfusion outcomes.

The AUSL-IRCCS of Reggio Emilia Transfusion Unit operates in two blood donor centers. Plasmapheresis protocols and machines are identical in both centers, except for the final unit weight setting: 700 g in Center 1 and 720 g in Center 2. Within a wider study to assess the anticoagulant content in plasma units through proton nuclear magnetic resonance, we compared the efficiency of the two settings. We analyzed 215 and 100 consecutive samples from Centers 1 and 2, respectively. We collected processed blood volume, net plasma collected and anticoagulant volume in the plasma units. In our experience, setting the machine at 720 g instead of 700 g was associated with a small increase in plasma content of the final unit (only 4 mL), but implied an increase of more than 100 mL of the total processed blood and a higher amount of anticoagulant in the unit. On the contrary, the difference in donor's reinfused anticoagulant was negligible. Our findings come from an observational study suggesting that, in view of a minimal advantage in terms of collected net plasma, there might be relevant disadvantages for the donor in prolonging plasmapheresis over 700 g. Since observed differences may be attributed to confounding factors, we recommend always checking the marginal efficiency of the procedure when the balance target value of the setting is increased. Randomized cross-over studies are needed to find the optimal target weight for plasma units. These studies could also help defining personalized plasmapheresis procedures, thus further optimizing donor safety.

BACKGROUND: While patient blood management (PBM) principles are not specific to cancer patients, their application contains the pathophysiological premises that could also benefit this patient population. In this study, we assessed the effects of implementing a PBM bundle for cancer patients in the postoperative period. MATERIALS AND METHODS: The Azienda USL-IRCCS of Reggio Emilia implemented a two-step PBM bundle for the postoperative period of cancer patients hospitalised in the semi-intensive post-surgery (SIPO) ward. Step 1 included seminars and lessons specifically targeting SIPO personnel; Step 2 introduced Points of Care (POCs) for the continuous monitoring of haemoglobin (Radical7, Masimo Corp, Irvine, CA, USA). We conducted 3 audits on 600 cancer patients recruited between 2014 and 2017: Audit 1 on 200 patients before the application of our PBM bundle; Audit 2 after Step 1 on 200 patients; Audit 3 after Step 2 on 200 patients monitored with POCs. Red blood cell (RBC) transfusion appropriateness in the postoperative period was evaluated using the Italian Society of Transfusion Medicine and Immunohaematology (SIMTI) recommendations. RESULTS: RBC transfusion appropriateness in the postoperative period of cancer patients rose from 38% to 75% after seminars, and reached 79% after the introduction of POC. The mean number of RBC units each patient received remained unchanged after training sessions (1.8 units/patient) while the introduction of POCs saw a simultaneous decrease in the number of prescribed units (1.3 units/patient). DISCUSSION: Our PBM bundle positively impacted RBC transfusion appropriateness in postsurgical cancer patients, both in terms of quality and quantity. A structured PBM programme specifically dedicated to surgical oncology should cover the entire perioperative period and might further improve transfusion appropriateness in these patients. The publication of guidelines on the management of anaemia in surgical oncology should be a priority.

The upholding of red blood cells (RBC) quality and the removal of leukocytes are two essential issues in transfusion therapy. Leukodepletion provides optimum results, nonetheless there are cases where irradiation is recommended for some groups of hematological patients such as the ones with chronic graft-vs-host disease, congenital cellular immunodeficiency, and hematopoietic stem cell transplant recipients. The European guidelines suggest irradiation doses from 25 to 50 Gray (Gγ). We evaluated the effect of different prescribed doses (15 to 50 Gγ) of X-ray irradiation on fresh leukodepleted RBCs bags using a novel protocol that provides a controlled irradiation. Biochemical assays integrated with RBCs metabolome profile, assessed by nuclear magnetic resonance spectroscopy, were performed on RBC units supernatant, during 14 days storage. Metabolome analysis evidenced a direct correlation between concentration increase of three metabolites, glycine, glutamine and creatine, and irradiation dose. Higher doses (35 and 50 Gγ) effect on RBC mean corpuscular volume, hemolysis, and ammonia concentration are considerable after 7 and 14 days of storage. Our data show that irradiation with 50 Gγ should be avoided and we suggest that 35 Gγ should be the upper limit. Moreover, we suggest for leukodepleted RBCs units the irradiation with the prescribed dose of 15 Gγ, value at center of bag, and ranging between 13.35-15 Gγ, measured over the entire bag volume, may guarantee the same benefits of a 25 Gγ dose assuring, in addition, a better quality of RBCs.

Allogeneic peripheral blood-derived (PBS) serum eye drops have been largely used in the treatment of dry eye disease (DED). Recently, cord blood has emerged as an effective alternative serum source (cord blood serum, CBS), containing a higher amount of growth factors than PBS, it holds the promise of a better capability to stimulate corneal healing. However, the lack of a standardized method for preparation, dispensation, storage and a poor biochemical characterization still hamper the establishment of a clinical consensus. Here the metabolomes of the two different serum eye drop preparations were compared using proton nuclear magnetic resonance spectroscopy. We found that both PBS and CBS contained several organic compounds, the majority of them already detected in human tears and may be thereby considered lacrimal substitutes. Metabolites having in the multivariate statistical analysis Partial least squares discriminant analysis (PLS-DA) a VIP scores > 1.0 were considered to be significantly different. All the metabolites identified were found to have a p < 0.05 in the univariate analysis. CBS, in particular, showed the highest amount of choline, myo-inositol, glutamine, creatine and β-hydroxybutyrate. These evidences constitute relevant advances towards serum eye drops characterization and confirm that cord blood is a valid alternative source of serum eye drops.

BACKGROUND: Anticoagulant concentration in plasma units is extremely variable. Understanding the underlying causes of this variability could help personalise plasmapheresis procedures in order to optimise the risk-benefit ratio. We studied the association between anticoagulant solution A (usually ACD-A, Citrate Dextrose Solution A) volume in plasma units and donor characteristics to build a model to determine the needed weight of the final plasma unit to have an 80% probability of reaching 600 mL net plasma. MATERIALS AND METHODS: We experimentally measured ACD-A in 296 plasma units from an Italian blood donor centre, where machines are set for the collection of 700 g of plasma. Next, we built a statistical model to predict how the final volume of the unit should be set to obtain 50%, 80% or 90% probability of having at least 600 mL net plasma. RESULTS: ACD-A volume was associated with haemoglobin, total proteins and triglycerides. Donors with low haemoglobin reach an 80% probability of at least 600 mL net plasma with units of approximately 690 g, while 720 g are needed for donors with high haemoglobin levels. For total proteins and triglycerides, plasma units may vary within a range of ±20 g. DISCUSSION: Our model, based on easily measurable individual characteristics, makes it possible to customise plasmapheresis procedures by determining the blood volume to be processed for each donor. Tailored plasma donations might result in both a reduction in adverse events and an increase in the quality of collected plasma.

BACKGROUND: To date, the quantification of the anticoagulant (ACD-A) in plasma units has been based on theoretical calculations. An accurate quantification could help minimize the risks associated with plasmapheresis, given that the total ACD-A used during the procedure is distributed between the donor and the plasma unit. Our aim was to experimentally quantify the volume of ACD-A in units collected by plasmapheresis. STUDY DESIGN AND METHODS: We used proton nuclear magnetic resonance spectroscopy to measure the ACD-A volume in 295 plasma units collected by the Azienda USL-IRCCS of Reggio Emilia, Italy. We analyzed the determinants of the differences between estimated and measured ACD-A through multivariate regression models. RESULTS: The experimentally measured ACD-A in plasma units was variable, with 45% of the samples showing a discrepancy of more than 15 mL compared to the manufacturer's estimate. ACD-A was underestimated for higher density of the units (p < 0.0005); a weak association was also observed with triglycerides (underestimated for higher levels, p = 0.015) and sex (overestimated in females, p = 0.008), but our model explained only 35% of the individual variability. CONCLUSION: The manufacturer's algorithms do not accurately estimate the ACD-A in units collected by plasmapheresis. Donor-related characteristics may affect ACD-A distribution between donor and plasma unit, thereby explaining the discrepancies between estimate and measurement. Errors in the estimate of the ACD-A actually received by donors could hamper studies on dose-response relationship between anticoagulant and adverse reactions. Our work should stimulate research on tailored procedures aimed at minimizing the anticoagulant received by donors and increasing plasmapheresis safety.

Following the identification of a novel polycyclic scaffold, leading to the previously reported potent P-gp modulator 1, a small series of easily affordable derivatives bearing a properly selected nitrogen-containing but-2-ynyl side chain was now synthesized and tested to evaluate the MDR reverting activity on two different experimental models. All compounds proved not to be cytotoxic when tested alone and more potent chemosensitizers than the reference verapamil. Some of them showed remarkable effects in combination with doxorubicin, being able to induce apoptotic cell death due to their reverting activity. In particular, 2a and 2c could be regarded as non-toxic new potential chemosensitizers, being able to interfere with different ABC proteins. Moreover, the intrinsic cytotoxicity of compound 1 could broaden its employment as MDR modulator. These results also seem to confirm the polycyclic core of these compounds as a potential new pharmacophoric carrier in medicinal chemistry.

The synthesis of new substituted E -3-(3-indolylmethylene)1,3-dihydroindol-2-ones is reported. The antiproliferative activity was evaluated according to protocols available at the National Cancer Institute (NCI), Bethesda, MD. The action of the most active compound 10 was further investigated in HL-60 leukemia cells. Results obtained show that it causes a block in cell cycle progression, with cell arrest in the G2/M phase, associated with activation of apoptosis accompanied with increased oxidative stress and deregulation of the homeostasis of divalent cations, with significant increase in the cellular concentrations of free Ca 2+ and Mg 2+ .

Magnesium plays a pivotal role in energy metabolism and in the control of cell growth. While magnesium deprivation clearly shapes the behavior of normal and neoplastic cells, little is known on the role of this element in cell differentiation. Here we show that magnesium deficiency increases the transcription of multipotency markers and tissue-specific transcription factors in human adipose-derived mesenchymal stem cells exposed to a mixture of natural molecules, i.e., hyaluronic, butyric and retinoid acids, which tunes differentiation. We also demonstrate that magnesium deficiency accelerates the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells. We argue that magnesium deprivation generates a stressful condition that modulates stem cell plasticity and differentiation potential. These studies indicate that it is possible to remodel transcription in mesenchymal stem cells by lowering extracellular magnesium without the need for genetic manipulation, thus offering new hints for regenerative medicine applications.

The mechanism of action of the mitochondrial Mg channel MRS2 and its involvement in cell viability remain unclear. Deletion of MRS2 has been reported to abolish Mg influx into mitochondria, to induce functional defects in mitochondrial organelles, and to result in cell death. We evaluated whether MRS2 expression had an impact on total Mg cellular content by inducing the overexpression of MRS2 in HEK-293 cells. We observed a remarkable increase of total intracellular Mg concentration in cells overexpressing MRS2 compared with control cells. In order to investigate whether and in what manner the detected Mg increment was involved in the MRS2 influence on cell viability, we treated MRS2-overexpressing cells with two known apoptotic inducers. We found that cells overexpressing the MRS2 channel became less responsive to these pharmacological insults. Our experimental evidence indicates that the MRS2 channel controls overall intracellular Mg levels, the alteration of which might have a role in the molecular signaling leading to apoptotic cell death.

We have recently developed an optimised synthetic strategy for the preparation of chemosensors based on the diaza-18- crown-6 hydroxyquinoline structure. This flexible approach allows to obtain a variety of substituted derivatives with improved fluorescence, metal ion binding, uptake and localisation properties. In this context, we have focused our attention on the design of specific chemosensors for intracellular Zn2? ions, because of their important role in many biological processes. Interestingly, among them one in particular shows the properties of making it a promising candidate for effective Zn(II) ions detection in biological environments. It presents, in fact, a very low toxicity, Zn2+/Mg2+ selectivity and a very large fluorescence enhancement on complexation.

Background Alkaptonuria (AKU) is an ultra-rare disease associated to the lack of an enzyme involved in tyrosine catabolism. This deficiency results in the accumulation of homogentisic acid (HGA) in the form of ochronotic pigment in joint cartilage, leading to a severe arthropathy. Secondary amyloidosis has been also unequivocally assessed as a comorbidity of AKU arthropathy. Composition of ochronotic pigment and how it is structurally related to amyloid is still unknown. Methods We exploited Synchrotron Radiation Infrared and X-Ray Fluorescence microscopies in combination with conventional bio-assays and analytical tools to characterize chemical composition and morphology of AKU cartilage. Results We evinced that AKU cartilage is characterized by proteoglycans depletion, increased Sodium levels, accumulation of lipids in the peri-lacunar regions and amyloid formation. We also highlighted an increase of aromatic compounds and oxygen-containing species, depletion in overall Magnesium content (although localized in the peri-lacunar region) and the presence of calcium carbonate fragments in proximity of cartilage lacunae. Conclusions We highlighted common features between AKU and arthropathy, but also specific signatures of the disease, like presence of amyloids and peculiar calcifications. Our analyses provide a unified picture of AKU cartilage, shedding a new light on the disease and opening new perspectives. General significance Ochronotic pigment is a hallmark of AKU and responsible of tissue degeneration. Conventional bio-assays have not yet clarified its composition and its structural relationship with amyloids. The present work proposes new strategies for filling the aforementioned gap that encompass the integration of new analytical approaches with standardized analyses.

Intra-operative blood cell salvage (IOCS) is mainly avoided in onco surgery due to the suspicion that it could increase metastasis' risk. We simulated IOCS followed by leucodepletion: HCT116 (human colorectal cancer) cells were inoculated into packed red blood cells units, and their distribution was evaluated, step-by-step, by flow cytometry and immunohistochemistry. Most of HCT116 cells were lost during washing, and almost completely removed after filtration. IOCS plus leucodepletion could be of great advantage for oncological patients, where allogenic blood transfusion could influence tumour progression.

We report a method that allows a complete quantitative characterization of whole single cells, assessing the total amount of carbon, nitrogen, oxygen, sodium, and magnesium and providing submicrometer maps of element molar concentration, cell density, mass, and volume. This approach allows quantifying elements down to 10(6) atoms/mu m(3). This result was obtained by applying a multimodal fusion approach that combines synchrotron radiation microscopy techniques with off-line atomic force microscopy. The method proposed permits us to find the element concentration in addition to the mass fraction and provides a deeper and more complete knowledge of cell composition. We performed measurements on LoVo human colon cancer cells sensitive (LoVo-S) and resistant (LoVo-R) to doxorubicin. The comparison of LoVo-S and LoVo-R revealed different patterns in the maps of Mg concentration with higher values within the nucleus in LoVo-R and in the perinuclear region in LoVo-S cells. This feature was not so evident for the other elements, suggesting that Mg compartmentalization could be a significant trait of the drug-resistant cells.

The present study investigated the analytical capabilities of a new fluorescent chemosensor, named DCHQ5, a phenyl derivative belonging to the family of diaza-crown-hydroxyquinolines, for the quantitative assessment of total intracellular Mg content. The results obtained were compared to the analytical performances of DCHQ1 - the parent probe of the series which so far was the only suitable species for the quantitative assessment of the intracellular total magnesium and showed comparable results to atomic absorption spectroscopy. Different protocols were tested in several cell lines both by flow cytometry and by steady state fluorescence spectroscopy assays. The results obtained support the possibility to use DCHQ5 to accurately quantify the intracellular total Mg in much smaller samples than DCHQ1, also displaying an increased stable intracellular staining. These features, combined with the high fluorescence enhancement upon cation binding, and the possibility to be excited both in the UV and visible region, make DCHQ5 a valuable and versatile analytical tool for Mg assessment in biological samples.

A recent study showed the antitumor activity of a new indole-derivative – MM-67 – inducing mitochondria-mediated apoptosis and a decrease of intracellular magnesium (Mg) concentration in HT29 colon cancer cells. Aim of this work was to assess cellular Mg levels throughout MM-67-induced apoptosis from the early to the final stage of the process and to evaluate the correlation with mitochondrial membrane potential (ΔΨm) variations. All analysis were performed by flow cytometry: ΔΨm was assessed by using mitochondrial potential sensitive dye DiOC6, while free and total intracellular cation concentrations were assessed by using the commercial probe MagFluo4-AM (Kd=4.7 mM), and the new synthesized DCHQ5 (Kd=8.3 mM), respectively. Our results evidenced that the MM67 induced apoptosis is characterized by a direct correlation between ΔΨ and free intracellular Mg content variations.

The current status of the TwinMic beamline at Elettra synchrotron light source, that hosts the European twin X-ray microscopy station, is reported. The X-ray source, provided by a short hybrid undulator with source size and divergence intermediate between bending magnets and conventional undulators, is energy-tailored using a collimated plane-grating monochromator. The TwinMic spectromicroscopy experimental station combines scanning and full-field imaging in a single instrument, with contrast modes such as absorption, differential phase, interference and darkfield. The implementation of coherent diffractive imaging modalities and ptychography is ongoing. Typically, scanning transmission X-ray microscopy images are simultaneously collected in transmission and differential phase contrast and can be complemented by chemical and elemental analysis using across-absorption-edge imaging, X-ray absorption near-edge structure or low-energy X-ray fluorescence. The lateral resolutions depend on the particular imaging and contrast mode chosen. The TwinMic range of applications covers diverse research fields such as biology, biochemistry, medicine, pharmacology, environment, geochemistry, food, agriculture and materials science. They will be illustrated in the paper with representative results.

Elemental analysis of biological sample can give information about content and distribution of elements essential for human life or trace elements whose absence is the cause of abnormal biological function or development. However, biological systems contain an ensemble of cells with heterogeneous chemistry and elemental content; therefore, accurate characterization of samples with high cellular heterogeneity may only be achieved by analyzing single cells. Powerful methods in molecular biology are abundant, among them X-Ray microscopy based on synchrotron light source has gaining increasing attention thanks to its extremely sensitivity. However, reproducibility and repeatability of these measurements is one of the major obstacles in achieving a statistical significance in single cells population analysis. In this study, we compared the elemental content of human colon adenocarcinoma cells obtained by three distinct accesses to synchrotron radiation light.

In synchrotron facilities, imaging techniques are on high demand from the scientific community. Those related to X-ray microscopy are among the most prominent ones. Such techniques include scanning transmission x-ray microscopy (STXM), full-field transmission x-ray microscopy (TXM), and coherent diffraction imaging (CDI) which have a wide spectrum of applications ranging from clinical and biomedical sciences to nanotechnology and cultural heritage. Their advancement is achieved through specialisation and focused studies, often requiring dedicated beamline end-stations. On the other hand, scientific applications benefit from the combination of techniques in a complementary manner. Beamlines suitably designed to offer multiple techniques, instead of a single one, can host efficiently such combinatorial studies. In this paper, we present the diverse Soft X-ray microscopy techniques in use at the TwinMic beamline at Elettra Sincrotrone Trieste, namely, STXM combined with XRF spectroscopy, full-field TXM and Ptychography. We demonstrate the capabilities by examining two specific biological samples: U87MG cells exposed to CoFe2O4 nanoparticles and a stem section of a Solanum lycopersicum (tomato) plant. These specimens are representatives of a large sample class used in a wide range of scientific studies. The results show the potential that can be achieved in terms of imaging by accessing X-ray microscopy techniques during a single beamtime access in TwinMic.

The biological function of a chemical element in cells not only requires the determination of its intracellular quantity, but also the spatial distribution of its concentration. Different strategies can be employed to quantify and map the intracellular concentration of elements in single cells. The assessment of the intracellular elemental concentration, which is the relevant information, requires the measurement of cell volume. This challenging and demanding task requires combining different techniques allowing gathering of both morphological and compositional information on the same cell. Moreover, the need to analyse samples more similar to their natural state requires complex hardware equipment, and supplementary efforts in preparation protocols. Nevertheless, the response to the question: "where is it and how much?" is worth all these efforts. This review aims at providing an insight into the recent and most advanced techniques and strategies for quantifying and mapping chemical elements in single cells. We describe and discuss indirect detection techniques (label based) which make use of fluorescent dyes, and direct ones (label free), such as particle induced X-ray emission, proton backscattering spectrometry, scanning transmission ion spectrometry, nano-secondary ion mass spectrometry, X-ray fluorescence microscopy, complemented by X-ray imaging.

CNNM2 is associated with the regulation of serum Mg concentration, and when mutated, with severe familial hypomagnesemia. The function and cellular localization of CNNM2 and its isomorphs (Iso) remain controversial. The objective of this work was to examine the following: (1) the transcription-responsiveness of CNNM2 to Mg starvation, (2) the cellular localization of Iso1 and Iso2, (3) the ability of Iso1 and Iso2 to transport Mg(2+), and (4) the complex-forming ability and spectra of potential interactors of Iso1 and Iso2. The five main findings are as follows. (1) Mg-starvation induces CNNM2 overexpression that is markedly higher in JVM-13 cells (lymphoblasts) compared with Jurkat cells (T-lymphocytes). (2) Iso1 and Iso2 localize throughout various subcellular compartments in transgenic HEK293 cells overexpressing Iso1 or Iso2. (3) Iso1 and Iso2 do not transport Mg(2+) in an electrogenic or electroneutral mode in transgenic HEK293 cells overexpressing Iso1 or Iso2. (4) Both Iso1 and Iso2 form complexes of a higher molecular order. (5) The spectrum of potential interactors of Iso1 is ten times smaller than that of Iso2. We conclude that sensitivity of CNNM2 expression to extracellular Mg(2+) depletion depends on cell type. Iso1 and Iso2 exhibit a dispersed pattern of cellular distribution; thus, they are not exclusively integral to the cytoplasmic membrane. Iso1 and Iso2 are not Mg(2+) transporters per se. Both isomorphs form protein complexes, and divergent spectra of potential interactors of Iso1 and Iso2 indicate that each isomorph has a distinctive function. CNNM2 is therefore the first ever identified Mg(2+) homeostatic factor without being a Mg(2+) transporter per se.

e18793Background: Patient blood management (PBM) is an evidence-based, multidisciplinary and multimodal approach aiming to optimize the care of patients (pts) who might need blood transfusions.This...

Neoplastic cells accumulate magnesium, an event which provides selective advantages and is frequently associated with TRPM7overexpression. Little is known about magnesium homeostasis in drug-resistant cancer cells. Therefore, we used the colon cancer LoVo cell model and compared doxorubicin-resistant to sensitive cells. In resistant cells the concentration of total magnesium is higher while its influx capacity is lower than in sensitive cells. Accordingly, resistant cells express lower amounts of the TRPM6 and 7, both involved in magnesium transport. While decreased TRPM6 levels are due to transcriptional regulation, post-transcriptional events are involved in reducing the amounts of TRPM7. Indeed, the calpain inhibitor calpeptin markedly increases the levels of TRPM7 in resistant cells. In doxorubicin-sensitive cells, silencing TRPM7 shifts the phenotype to one more similar to resistant cells, since in these cells silencing TRPM7 significantly decreases the influx of magnesium, increases its intracellular concentration and increases resistance to doxorubicin. On the other hand, calpain inhibition upregulates TRPM7, decreases intracellular magnesium and enhances the sensitivity to doxorubicin of resistant LoVo cells. We conclude that in LoVo cells drug resistance is associated with alteration of magnesium homeostasis through modulation of TRPM7. Our data suggest that TRPM7 expression may be an additional undisclosed player in chemoresistance.

Osteosarcoma (OS) is the most common primary malignant tumor of bone, occurring most frequently in children and adolescents. The mechanism of formation and development of OS have been studied for a long time. Tumor suppressor pathway governed by p53 gene are known to be involved in the pathogenesis of osteosarcoma. Moreover, loss of wild-type p53 activity is thought to be a major predictor of failure to respond to chemotherapy in various human cancers. In previous studies, we described the activity of a new indole derivative, NSC743420, belonging to the tubulin inhibitors family, capable to induce apoptosis and arrest of the cell cycle in the G2/M phase of various cancer cell lines. However, this molecule has never been tested on OS cell line. Here we address the activity of NSC743420 by examine whether differences in the p53 status could influence its effects on cell proliferation and death of OS cells. In particular, we compared the effect of the tested molecule on p53-wild type and p53-silenced U2OS cells, and on SaOS2 cell line, which is null for p53. Our results demonstrated that NSC743420 reduces OS cell proliferation by p53-dependent and p53-independent mechanisms. In particular, the molecule induces proliferative arrest that culminate to apoptosis in SaOS2 p53-null cells, while it brings a cytostatic and differentiating effect in U2OS cells, characterized by the cell cycle arrest in G0/G1 phase and increased alkaline phosphatase activity.

The differing bioavailability of magnesium salts remains an open question, both at the cellular and systemic level. However, this issue is relevant for identifying the most effective magnesium supplement. We compared the effects of three widely used magnesium salts: MgSO4, MgCl2 and Mg pidolate, on the proliferation of four human cell types: promyelocytic leukaemia HL60, osteoblast-like Saos-2 and U-2 OS, and endothelial cells from the umbilical vein. The three magnesium salts had no effect on endothelial and leukemic cell growth, but magnesium pidolate impaired cell growth in osteoblast-like cells. In particular, in Saos-2 cells, 1 mM pidolate induced a slight accumulation of cells in the G0/G1 phase of the cell cycle and, in parallel, an early rise in intracellular calcium and a late decrease in intracellular magnesium content. Interestingly, when cultured in 5 mM magnesium pidolate, Saos-2 cells grew as fast as the controls. Moreover, intracellular magnesium and calcium concentrations did not vary. These results suggest a lower bioavailability of magnesium pidolate in osteoblast-like cells.

Magnesium (Mg) is essential for biological processes, but its cellular homeostasis has not been thoroughly elucidated, mainly because of the inadequacy of the available techniques to map intracellular Mg distribution. Recently, particular interest has been raised by a new family of fluorescent probes, diaza-18-crown-hydroxyquinoline (DCHQ), that shows remarkably high affinity and specificity for Mg, thus permitting the detection of the total intracellular Mg. The data obtained by fluori- metric and cytofluorimetric assays performed with DCHQ5 are in good agreement with atomic absorption spectroscopy, confirming that DCHQ5 probe allows both qualitative and quantitative determination of total intracellular Mg.

A newly synthesized indole-derivative is able to induce cytostatic and cytotoxic effects in the colon cancer cells HT29, effecting apoptosis by activation of an intrinsic pathway. Magnesium is involved in both cell growth and apoptosis even though its role in the latter process is not well defined. The aims of this work were: firstly, to verify if magnesium content is related to the proliferative rate in HT29 cells; secondly, to assess the involvement of the cation in mitochondria-mediated apoptosis triggered by the new antiproliferative molecule. The effects of the indole-derivative in treated cells included cell-cycle arrest in the G2/M phase, and apoptotic death confirmed by release of cytochrome c from the mitochondrial compartment. Moreover, we demonstrated that the basal content of magnesium in HT29 cells inversely correlates with cell saturation density. In addition, a decrease in both free and intracellular total magnesium concentration was observed along with the induced apoptosis. Taken together, these data suggest that magnesium participates in the complex signaling network of cell proliferation and apoptosis.

We present here a new methodology for quantitative mapping of light elements in cells, based on combination of compositional and morphological information, derived respectively by X-ray Fluorescence Microscopy (XRFM), Atomic Force Microscopy and Scanning Transmission X-ray Microscopy (STXM). Since XRFM of light elements (carbon, nitrogen, oxygen, sodium and magnesium), are strongly influenced by self-absorption, we developed an algorithm to correct for this effect, using the morphological and structural information provided by AFM and STXM. Finally, the corrected distributions have been obtained, thus allowing quantitative mapping.